Prognostic biomarkers for ttk inhibitor chemotherapy

ABSTRACT

A method for identifying a tumor that is susceptible to treatment with a TTK inhibitor, including: a) providing a sample of a tumor; b) determining the presence of a mutated CTNNB1 gene in the sample, wherein the mutation is located in exon 3 of CTNNB1 and the presence of a mutated CTNNB1 gene indicates the tumor is susceptible to treatment with a TTK inhibitor. Alternatively, step b) is replaced by the step of determining the presence of a mutated CTNNB1 protein in the sample, wherein the mutation is located in exon 3 of CTNNB1 and the presence of a mutated CTNNB1 protein indicates the tumor is susceptible to treatment with a TTK inhibitor. In a further alternative, step b) includes determining an altered expression of a CTNNB1 regulated gene, whereby an altered expression of a CTNNB1 regulated gene indicates the tumor is susceptible to treatment with a TTK inhibitor.

FIELD OF THE INVENTION

The present invention relates to methods to detect the mutant status ofthe CTNNB1 gene or CTNNB1 protein or the altered expression of a CTNNB1regulated gene to identify tumors that are susceptible to anticancertherapy with a TTK inhibitor. The present invention also relates tomethods to predict the outcome, or disease progression, of cancers thatare treated with a TTK inhibitor, by detection of the mutant status ofthe CTNNB1 gene or CTNNB1 protein or the altered expression of a CTNNB1regulated gene

BACKGROUND OF THE INVENTION

Targeted therapies bring great benefit to cancer patients because theycan improve survival rates with fewer side effects than traditional,less selective cytotoxic drugs. Small molecule inhibitors of proteinkinases are a prime example of the success of targeted therapy: many ofthese inhibitors exploit unique features of tumor cells, permittingcancer specificity while having limited effects on healthy cells. Aclassic example of a targeted therapy is the use of tyrosine kinaseinhibitors and antibodies in breast cancer patients with amplificationor overexpression of the HER² gene (Higgins, M. J., and Baselga, J., J.Clin. Invest. 121: 3797; 2011).

To determine whether it is likely that a patient will respond to acertain targeted therapy, it is important to determine the status andpresence of biomarkers that correlate with drug sensitivity in specimensof the patient's tumor, before the start of treatment.

The protein kinase TTK (EC 2.7.12.1), commonly referred to as Mps1, is acomponent of the spindle assembly checkpoint (SAC), a surveillancemechanism that ensures the fidelity of chromosome segregation (Liu, X.,and Winey, M., Annu. Rev. Biochem. 81: 561; 2012). Defects in SACfunctioning can lead to chromosome segregation errors by allowingmitotic exit in the presence of unattached kinetochores. Complete lossof SAC function is lethal in mice (Baker, D. J., et al., Curr. Opin.Cell Biol. 17, 583; 2005) and incompatible with the viability of humancell lines (Michel, L., et al. Proc. Natl. Acad. Sci. USA 101, 4459;2004; Kops G. J., et al. Proc. Natl. Acad. Sci. USA 101, 8699; 2004).TTK mRNA levels are elevated in various human cancers, including breast,thyroid papillary carcinoma, hepatocellular carcinoma, pancreatic ductaladenocarcinoma, glioma, gastric, bronchogenic, and lung (Daniel, J., etal. Proc. Natl. Acad. Sci. USA 108: 5384; 2011; Maire, V., et al., PLoSONE 8(5) e63712; 2013; Kilpinen, S., et al., PLoS ONE 5(12), e15068;2010; Landi, M. T., et al., PLoS ONE 3(2) e1651; 2008; Liang, X. D., etal. PLoS ONE 9(6), e97739; 2014; Mills, G. B., et al. J. Biol. Chem.267: 16000; 1992; Mir, S. E., et al., Cancer Cell 18: 244; 2010;Salvatore, G., et al., Cancer Res. 67: 10148; 2007: Slee, R. B., et al.,Mol. Cancer Ther. 13: 307; 2014; Tannous, B. A., et al., J. Natl. CancerInst. 105: 1322; 2013; Yuan, B., et al., Clin. Cancer Res. 12: 405;2006). Therefore, chemical compounds that inhibit the activity of TTKare useful in the treatment of a variety of cancers. These compounds maybe applied as single agents, or in combination with other anti-canceragents.

Different compounds have been disclosed which show an inhibitory effecton TTK. AstraZeneca UK Ltd. disclosed 2-anilinopurin-8-ones asinhibitors of TTK in WO2009/024824 A1. In WO2011/013729 A1, fusedimidazoles, and in WO2011/016472 A1 pyridine and pyrimidine derivativesare disclosed as inhibitors of TTK by Oncotherapy Science Inc. Indazolesfor inhibition of TTK have been disclosed by University Health Networkin WO2011/123937 A1, WO2013/053051 A1 and WO2014/056083 A1. Dana FarberCancer Institute disclosed pyrimido-diazepinones as inhibitors of TTK inWO2010/080712 A1. In WO2009/156315 A1, pyrazolo-quinazolines, inWO2012/101029 A1 tricyclic derivatives, in WO2010/108921 A1,N-aryl-2-(2-arylaminopyrimidin-4-yl)pyrrol-4-carboxamides, inWO2012/013557 A1, isoxazolo-quinazolines, in WO2012/101032 A1, tricyclicpyrrolo derivatives and in WO2012/139930 A1, pyrazolyl-pyrimidines aredisclosed as inhibitors of TTK by Nerviano Medical Sciences S.R.L.

Myriad Pharmaceuticals Inc. disclosed purines as inhibitors of TTK inWO2010/111406 A2. Furthermore, Cancer Research Technology Ltd. disclosedpyrrolopyridineamino derivatives in WO2012/123745 A1 and bicycles inWO2014/037750 A1 and in WO2014/037751 A1 as inhibitors of TTK.

In WO2010/124826 A1, imidazoquinoxalines, in WO2011/026579 A1,aminoquinoxalines, in WO2011/063907 A1, WO2011/063908 A1, WO2011/064328A1, WO2011/157688 A1, WO2012/143329 A1, WO2014/009219 A1, WO2014/195274A1, WO2014/195276 A1 and WO2014/198647 A1, triazolopyridines, inWO2012/136531 A1, imidazopyridines, in WO2012/130905 A1, substitutedbenzimidazoles, in WO2012/032031 A1, WO2013/135612 A1 and WO2014/131739A1, imidazopyridazines, in WO2011/113862 A1, WO2011/151259 A1,WO2012/080228 A1, WO2012/080229 A1, WO2012/080230 A1, WO2012/080232 A1,WO2012/080234 A1 and WO 2012/080236 A1, imidazopyrazines arerespectively disclosed as inhibitors of TTK by Bayer Schering PharmaA.G.

Representative compounds of the different chemical classes have beeninvestigated in cell proliferation assays with different human cancercell lines. A representative TTK inhibitor of the imidazo-pyrazines,Mps-BAY2b, was shown to inhibit the proliferation of twenty-seven humancancer cell lines from different tumor origins with an IC₅₀ of 160 nM to4.3 μM (Jemaa, M., et al., Cell Death Different. 20: 1532; 2013); nocorrelation was found between the response and the pattern of genomicinstability, the activity of several proteins relevant for oncogenesis,or the functionality of the SAC.

NMS-P715, a representative of the pyrazolo-quinazoline class, inhibitedthe proliferation of a wide range of cell lines in a panel of 127 cancercell lines (Colombo, R., et al., Cancer Res. 70: 10255; 2010); IC₅₀swere close to 1 μM or higher and there was no correlation observedbetween anti-proliferative effects and cellular doubling time.

MPI-04079605, a TTK inhibitor disclosed by Myriad, was shown to inhibitthe growth of fourteen human cancer cell lines from different tumororigins, but only after prolonged incubation time (Tardif, K. D., etal., Mol. Cancer Res. 10: 2267; 2011).

CCT251455, a representative of the 1H-pyrrolo[2,3-c]pyridine class,inhibited the proliferation of HCT116 cells with a GIso of 160 nM (Naud,S., et al., J. Med. Chem. 56: 10045; 2013).

An imidazo[1,2-b]pyridazine-based TTK inhibitor disclosed by Shionogi,was shown to inhibit the proliferation of fourteen human cancer celllines from different tumor origins with an IC₅₀ of 3.3 nM to 320 nM(Kusakabe, K., et al., J. Med. Chem. 58: 1716; 2015); CFI-401870, arepresentative of the indazoles, inhibited the proliferation of a widerange of cell lines in a panel of 22 cancer cell lines (Liu, Y., et al.,J. Med. Chem. 58: ASAP; 2015) with GI₅₀s of 8 nM to 70 nM.

Whereas in the above cited profiling experiments, different cancer celllines showed different relative sensitivities for TTK inhibitors, nogenomic or other markers were identified that correlated withsensitivity to TTK inhibitors.

Several TTK inhibitors of the above mentioned chemical classes have beenshown to reduce growth of xenografts in mouse models of melanoma(Colombo, R., et al.), colorectal carcinoma (Jemaa, M., et al.; Tardif,et al.; Laufer, R., et al., Bioorg. Med. Chem. 22: 4968; 2014), cervicalcarcinoma (Jemaa, M., et al.) and glioblastoma cells (Tannous, B. A. etal.), demonstrating the potential use of TTK inhibitors in treatment ofvarious cancers.

In view of the broad activity of TTK inhibitors in many different celllines and tumor types, there is a clear need for biomarkers that can beused to predict which cancers are most likely to respond tochemotherapeutic treatment with a TTK inhibitor. Such a prognostic drugsensitivity biomarker can be used to identify the most optimal patientpopulation to the application of drug therapy with a TTK inhibitor, orcan be used to predict the progression, or outcome of disease treatedwith a TTK inhibitor.

STATEMENT OF THE INVENTION

In accordance with a first aspect of the present invention there isprovided a method as defined in claim 1 appended hereto.

The present inventors have surprisingly observed that cancer cells thatharbor mutations in the CTNNB1 gene (HUGO name: CTNNB1) are moresensitive to TTK inhibitors than normal cells or cancer cells that donot express mutant CTNNB1 (CTNNB1 proficient cells).

The CTNNB1 gene encodes a dual function protein, p-catenin, whichregulates the coordination of cell adhesion and regulates genetranscription in the Wnt signaling pathway (Logan, C. Y., and Nusse, R.,Annu. Rev. Cell. Dev. Biol. 896: 1998; 2004). Mutations in the CTNNB1gene have been found in many cancers, including colorectal (Morin, P. J.et al., Science 275: 1787; 1997; Iwao, K., et al., Cancer Res. 58: 1021;1998; Sparks, A. B., et al. Cancer Res. 58: 1130; 1998), andhepatocellular carcinoma (Miyoshi, Y., et al., Cancer Res. 58: 2524;1998; Chen, Y. W., et al., Hepatology 36: 927; 2002), melanoma(Rubinfeld, B., et al., Science 275: 1790; 1997), medulloblastoma(Zurrawel, R. H., et al. Cancer Res. 58: 896; 1998), lung (Shigemitsu,K., et al., Oncogene 20: 4249; 2001), endometrial (Fukuchi, T., et al.,Cancer Res. 58: 3526; 1998; Liu, Y., et al., J. Natl. Canc. Inst.106(9); 2014), ovarium (Palacios, J., and Gamallo, C., Cancer Res. 58:1344; 1998) and prostate cancer (Voeller, H. J., and Gelmann, E. P.,Cancer Res. 58, 2520; 1998).

The activity of β-catenin is regulated by phosphorylation at serine andthreonine residues by the protein kinases glycogen synthase kinase 33(JGSK31) and casein kinase I (CKI), followed by ubiquitination anddegradation by the proteasome (Liu, C., et al. Cell 108, 837; 2002).Mutations in the CTNNB1 gene resulting in deletion or substitution ofone or more of these serine or threonine residues impairsphosphorylation and degradation, resulting in an overactive β-catenin,and uncontrolled cell growth (Morin, P. J. et al., Science 275: 1787;1997; Liu, C., et al.).

The present invention provides methods to determine the mutant status ofCTNNB1 in tumor derived materials, to determine the susceptibility ofsaid tumors to anticancer therapy with a TTK inhibitor. The presentinvention also provides methods to determine the mutant status of CTNNB1to monitor the effectiveness of therapy of proliferative disease with aTTK inhibitor, or to predict the outcome of cancers that are treatedwith a TTK inhibitor.

The analysis of the mutant status of CTNNB1 may be performed incombination with analyses of the mutant status or expression of othergenes and/or proteins, or may be confined to an analysis of only CTNNB1gene status.

The present invention constitutes a diagnostic method. However, themethod is not performed directly on the human or animal body. Thediagnostic method may be performed in a laboratory, but provides resultsthat allow a physician to make an accurate prognosis of diseaseprogression in a cancer patient, particularly with respect to whether apatient is likely to respond to chemotherapy with a TTK inhibitor,applied either as a single agent, or in combination with othertherapeutic agents or radiotherapy.

More specifically, the present invention provides methods to determinethe status of oncogenic CTNNB1 mutations in tumor derived materials todetermine the susceptibility of said tumors in anti-cancer therapy witha TTK inhibitor as defined in Formulas I-VIII detailed herein.

Many different mutations in β-catenin have been observed in cancerpatients, and these have been categorized in databases such as COSMIC(http://cancer.sanger.ac.uk/cancergenome/projects/cosmic/). Theexpression of CTNNB1 mutations in human cancers is reported in TheCancer Genome Atlas, which can be accessed athttp://www.cancerenome.nih.gov.

A link to the CTNNB1 nucleic acid and protein sequence can be found athttp://www.genenames.org/cgi-bin/ene_symbol_report?hgnc_id=2514 thedisclosure of which is herein incorporated by reference. The proteinsequence and amino acid numbering of CTNNB1 is also given in FIG. 1appended hereto.

Exon 3 of CTNNB1 contains a hot spot of mutations that affect theability of kinases to phosphorylate β-catenin (Morin, P. J. et al.,1997). The lack of this phosphorylation results in β-cateninaccumulation in the nucleus (Liu, C. et al., 2002). More specifically,mutation or deletion of the serine residues at positions 33, 37 or 45,or mutation or deletion of the threonine residue at position 41 alterthe GSK33 phosphorylation motifs which participate in the degradation ofβ-catenin (Rubinfeld, B., et al.; Morin, P. J., et al.). Consequently,these mutations result in increased oncogenic signaling (Rubinfeld, B.,et al.; Morin, P. J., et al.).

In accordance with a further aspect of the invention there is provided amethod according to claims 15 to 18 appended hereto. Specifically, amethod is described to determine whether a chemical compound is a TTKinhibitor, said method comprising the steps of: a) Providing first andsecond mammalian cell lines, wherein the first cell line isCTNNB1-mutated and the second cell line is CTNNB1 proficient; b)Contacting said first and second cell lines with a first candidatecompound; and, c) Determining by assay the inhibition of cellproliferation of said first and second cell lines. In an importantvariant of this method, steps b) and c) as mentioned above are repeatedwith a second candidate compound and a selection of candidate compoundis made based on the activity of the respective candidate compounds inthe assay with said first cell line.

In an embodiment, the first and second cell lines used in this methodmay be cancer cell lines. In an alternative embodiment, the first andsecond cell lines may be isogenic cell lines.

The present inventors have surprisingly observed that expression ofthree of the mutations described above correlates with increasedsusceptibility of cancer cells to chemical inhibitors of TTK. Therefore,detection of the mutant status of the CTNNB1 gene at serine 33,threonine 41, or serine 45 can be used to determine the susceptibilityof tumors for treatment with TTK inhibitors.

BRIEF DESCRIPTION OF THE FIGURES

The invention will be described with reference to the appended figuresin which:

FIG. 1 is the protein sequence and amino acid numbering of CTNNB1(β-catenin) (UniProt code P35222). Mutation or deletion of theunderlined serine (S) or threonine (T) residues at positions 33, 37, 41and 45 alter the phosphorylation and degradation of 3-catenin(Rubinfeld, B., et al.; Morin, P. J., et al.).

FIG. 2 represents the volcano plots of cellular profiling in 66 cancercell lines for Examples 5, 8, 9, 12, 13, 17 and 20.

For completeness, a volcano plot is a graphical representation of ananalysis of variance (Anova) of the association of cancer gene mutationspresent in cell lines and the response of these cell lines inproliferation assays with compounds. The volcano plot shows the averageIC₅₀ shift between mutant and non-mutant cell lines (x-axis) and thesignificance from the Anova test (y-axis). Significance was correctedfor multiple-testing and all associations above the threshold level(dotted line) are filled in black. Areas of circles are proportionalwith the number of cell lines carrying mutations. The cancer cell linesused in the drug sensitivity analysis are listed in Table 1 hereinbelow.

DETAILED DESCRIPTION OF THE INVENTION

Methods of obtaining a sample of a tumor for analysis are well known inthe art and require no specific elucidation here. The mutant status ofthe CTNNB1 gene of a tumor from an individual with cancer can bedetermined by analyzing the DNA sequence of a sample of the tumor, andcomparing the tumor DNA sequence with that in healthy tissue, or withthe ‘wild-type’ CTNNB1 sequence, referred to in the UniProt data base asP35222, and displayed in FIG. 1. A DNA sample may be taken directly froma tumor biopsy, or may be derived from circulating tumor DNA (Diaz, L.A., et al., Nature 486: 537; 2012). The mutant status of the CTNNB1 genemay also be determined by sequencing of the mRNA of the tumor sample, ormay be determined indirectly by analysis of the amino acid sequence ofβ-catenin, or by determination of the phosphorylation status ofβ-catenin using specific antibodies.

As the mutations affect the degradation of β-catenin, they affect thetotal cellular levels of 3-catenin and the amount β-catenin in thenucleus. Therefore, the mutant status of the CTNNB1 gene may also bedetermined indirectly by determining total or nuclear β-catenin levelsin tumor cells.

Alternatively, the mutant status of CTNNB1 may be determined byanalyzing the expression of genes that are regulated by β-catenin. Thedetection of β-catenin-regulated genes may be determined by extractingRNA from a sample of a tumor and measuring gene expression usingreverse-transcriptase polymerase chain reaction (RT-PCR) or usingmicroarray analysis. Many genes regulated by β-catenin have beendescribed, and include Axin2 (Yan, D. et al., Proc. Natl. Acad. Sci. USA98: 14973; 2001), c-myc (He, T. C. et al., Science 281: 1509; 1998) andLGR5 (Barker, N. et al., Nature 499: 1003; 2007). A comprehensive listof β-catenin-regulated genes can be found at the Wnt home page(http://web.stanford.edu/group/nusselab/cgi-bin/wnt/targetgenes) and inscientific articles (Willert, J. et al., BMC Dev. Biol. 2:8; van deWetering, M et al., Cell 111: 241; 2002).

The expression of β-catenin-regulated genes may also be determined atthe protein level, using specific antibodies or mass-spectroscopy-basedmethods. Since several β-catenin-regulated genes are oncogenes, themutant status of CTNNB1 can also be determined by measuring oncogenicsignaling.

Examples of inhibitors of TTK are chemical compounds belonging to theclass of (5,6-dihydro)pyrimido[4,5-e]indolizines according to Formula Ior pharmaceutically acceptable salts thereof.

wherein,

-   R¹ is selected from the group consisting of:

-   R¹¹ is H, halogen, (1-2C)alkyl, (2-3C)alkenyl, (2-3C)alkynyl,    (1-2C)alkoxy or OC₂H₃, all alkyl and alkoxy groups optionally being    substituted with one or more halogen;-   R¹² is H, halogen, (1-2C)alkyl or (1-2C)alkoxy;-   R¹³ is R¹³¹CH₂, R¹³²O, R¹³³R¹³⁴N, R¹³⁵C(O), R¹³⁶S, R¹³⁶S(O),    R¹³⁶S(O)(NH), R¹³⁷SO₂, (2-7C)heterocycloalkyl, or (1-5C)heteroaryl    each heterocycloalkyl or heteroaryl optionally being substituted    with (1-2C)alkyl, fluoro, hydroxyl, oxo, (1-2C)alkoxy,    (1-6C)alkylcarbonyl, (1-6C)alkylsulfonyl, (1-5C)alkoxycarbonyl,    (1-6C)alkylaminocarbonyl, (3-6C)cycloalkylcarbonyl,    (2-7C)heterocycloalkylcarbonyl or di[(1-2C)alkyl]amino, each    alkylcarbonyl, alkylsulfonyl, alkoxycarbonyl, alkylaminocarbonyl,    cycloalkylcarbonyl or heterocycloalkylcarbonyl optionally being    substituted with (1-2C)alkyl, fluoro, hydroxyl, cyano, oxo or    (1-2C)alkoxy;-   R¹³¹ is (1-6C)alkylcarbonylamino, (3-6C)cycloalkylcarbonylamino or    (2-7C)heterocycloalkylcarbonylamino each optionally substituted with    one or more groups selected from (1-2C)alkyl, fluoro, hydroxyl or    (1-2C)alkoxy;-   R¹³² is (1-6C)alkyl, (3-6C)cycloalkyl, (2-7C)heterocycloalkyl,    (6-10C)aryl or (1-5C)heteroraryl each optionally substituted with    one or more groups selected from (1-2C)alkyl, halogen, hydroxyl,    (1-2C)alkoxy, di[(1-2C)alkyl]amino or (2-7C)heterocycloalkyl;-   R¹³³ is (1-6C)alkyl, (3-6C)cycloalkyl, (2-7C)heterocycloalkyl    (1-6C)alkylcarbonyl, (1-5C)alkoxycarbonyl, (3-6C)cycloalkylcarbonyl    or (2-7C)heterocycloalkylcarbonyl, each optionally substituted with    one or more groups selected from (1-2C)alkyl, halogen, hydroxyl or    (1-2C)alkoxy, di[(1-2C)alkyl]amino or (2-7C)heterocycloalkyl;-   R¹³⁴ is hydrogen or (1-2C)alkyl;-   R¹³⁵ is (2-7C)heterocycloalkyl, (1-6C)alkylamino,    di[(1-6C)alkyl]amino, (2-7C)heterocycloalkylamino or    (3-6C)cycloalkylamino each optionally substituted with one or more    groups selected from (1-2C)alkyl, fluoro, hydroxyl, (1-2C)alkoxy,    di[(1-2C)alkyl]amino, (2-7C)heterocycloalkyl, oxo, cyano or amino;-   R¹³⁶ is (1-6C)alkyl, (3-6C)cycloalkyl, (2-7C)heterocycloalkyl each    optionally substituted with one or more groups selected from    (1-2C)alkyl, fluoro, hydroxyl or (1-2C)alkoxy;-   R¹³⁷ is (1-6C)alkyl, (3-6C)cycloalkyl, (2-7C)heterocycloalkyl,    (1-6C)alkylamino, di[(1-6C)alkyl]amino, (2-7C)heterocycloalkylamino    or (3-6C)cycloalkylamino, each optionally substituted with one or    more groups selected from (1-2C)alkyl, fluoro, hydroxyl or    (1-2C)alkoxy;-   R¹⁴ is H, halogen, (1-2C)alkyl or (1-2C)alkoxy; and-   R^(is) is H, halogen.

In the above Formula I, R² is selected from the group consisting of:

-   R²¹ is H, halogen, (1-3C)alkyl, (1-2C)alkoxy, hydroxy(1-2C)alkyl,    (3-4C)cycloalkyl, (2-3C)alkenyl or cyano;-   R²² is H, halogen, (1-2C)alkyl or (1-2C)alkoxy;-   R²³ is H, halogen, (1-2C)alkyl, (1-2C)alkoxy, cyano or hydroxy;-   R²⁴ is H, halogen, (1-2C)alkyl or (1-2C)alkoxy;-   R²⁵ is H, halogen, (1-3C)alkyl, (1-2C)alkoxy, hydroxy(1-2C)alkyl,    (3-4C)cycloalkyl, (2-3C)alkenyl or cyano;-   R²⁶ is H, (1-6C)alkyl, (3-6C)cycloalkyl, (2-5C)heterocycloalkyl,    (1-2C)alkoxy[(2-4C)alkoxy]_(n)(1-6C)alkyl, wherein n represents an    integer of 1, 2, 3 or 4, all alkyl, heterocycloalkyl and    (1-2C)alkoxy[(2-4C)alkoxy]_(n)(1-6C)alkyl groups optionally    substituted with one or more groups selected from (1-2C)alkyl,    (1-2C)alkoxy, hydroxyl, oxo, amino, (3-6C)cycloalkyl,    di[(1-2C)alkyl]amino or (2-5C)heterocycloalkyl.

In the above Formula I only one of R²¹ and R²⁵ in R² can be H.

Other examples of known TTK inhibitors are chemical compounds belongingto the class of pyrazolo-quinazolines according to Formula II orpharmaceutically acceptable salts thereof as described in WO2009/156315A1.

wherein,

-   -   R¹ and R³ are independently selected from the group consisting        of (6-10C)aryl and (1-5C)heteroaryl, wherein both groups        optionally can be substituted;    -   R² is selected from the group consisting of (1-6C)alkyl and        (2-6C)alkenyl, wherein both groups optionally can be        substituted;    -   R⁴ is selected from the group consisting of hydrogen and        (1-6C)alkyl, wherein both groups optionally can be substituted;    -   R⁵ and R⁶ are independently hydrogen or methyl.

Other, known TTK inhibitors are chemical compounds belonging to theclass of imidazo-pyrazines according to Formula III or pharmaceuticallyacceptable salts thereof as described in WO2011/013729 A1, WO2011/113862A1, WO2011/151259 A1, WO2012/080228 A1, WO2012/080229 A1, WO2012/080230A1, WO2012/080232 A1, WO2012/080234 A1 and WO 2012/080236 A1.

wherein,

-   -   R¹ is selected from the group consisting of (1-6C)alkyl,        halo(1-6C)alkyl, HO-(1-6C)alkyl, H₂N-(1-6C)alkyl,        cyano(1-6C)alkyl, (1-6C)alkoxy(1-6C)alkyl, (2-6C)alkenyl,        (2-6C)alkynyl, (3-6C)cycloalkyl, (3-7C)heterocycloalkyl,        (6-10C)aryl and (1-5C)heteroaryl, wherein said groups optionally        can be substituted;    -   R² is selected from the group consisting of (6-10C)aryl and        (1-9C)heteroaryl, wherein both groups optionally can be        substituted;    -   R³ is selected from the group consisting of: (1-6C)alkyl,        —(CH₂)_(n)-(3-7C) heterocycloalkyl),        —(CH₂)_(n)-(4-8C)heterocycloalkenyl), (3-7C)heterocycloalkyl,        (6-10C)aryl, (1-9C)heteroaryl, —(CH₂)_(n)-(6-10C)aryl,        —O-(6-10C)aryl, —C(═O)N and cyano, wherein said groups can be        substituted and further wherein n is an integer of 0, 1 or 2.

Another example of known TTK inhibitors are chemical compounds belongingto the class of purines according to Formula IV or pharmaceuticallyacceptable salts thereof as described in WO2010/111406 A1.

wherein,

-   -   R¹ is selected from the group consisting of (3-6C)cycloalkyl and        (3-7C)heterocycloalkyl, wherein said groups optionally can be        substituted; and,    -   R² is selected from the group consisting of:        -   a) (6-10C)aryl, and,        -   b) (1-5C)heteroaryl,    -   wherein both groups optionally can be substituted.

Yet, other known TTK inhibitors are chemical compounds belonging to theclass of imidazopyridazines according to Formula V or pharmaceuticallyacceptable salts thereof as described in WO2011/013729 A1, WO2012/032031A1, WO2013/135612 A1 and WO2014/131739 A1.

wherein,

-   -   R¹ is selected from the group consisting of hydrogen,        (1-6C)alkyl, halo(1-6C)alkyl, HO(1-6C)alkyl, (3-6C)cycloalkyl,        (3-7C)heterocycloalkyl and (1-5C)heteroaryl, wherein said groups        optionally can be substituted;    -   R² is (6-10C)aryl or (1-9C)heteroaryl, each of which may be        optionally substituted;    -   R³ is selected from the group consisting of X-(6-10C)aryl or        X-(1-9C)heteroaryl, wherein both groups optionally can be        substituted, wherein X represents S(═O)_(p), O, NR⁴, CR^(4a)R⁴b,        C═CR^(4a)R^(4b) and further wherein p is an integer of 0, 1, 2;    -   R⁴, R^(4a), R^(4b) represent independently from each other a        hydrogen atom or (1-6C)alkyl.

Other, known TTK inhibitors are chemical compounds belonging to theclass of triazolopyridines according to Formula VI or pharmaceuticallyacceptable salts thereof as described in WO2011/063907 A1, WO2011/063908A1, WO2011/064328 A1, WO2011/157688 A1, WO2012/143329 A1, WO2014/009219A1, WO2014/195274 A1, WO2014/195276 and WO2014/198647 A1.

wherein,

-   -   R¹ represents a phenyl group, a pyridyl group or an indolyl        group wherein said groups can optionally be substituted;    -   R² represents a phenyl group, a pyridyl group or a pyrimidyl        group wherein said groups can optionally substituted;    -   R³ represents a group selected from: hydrogen or        —C(═O)—O—(CR⁷R⁸)—O—C(═O)—R⁴ wherein R⁴ represents a group        selected from: (1-6C)alkyl, substituted one or more times,        identically or differentially, with a group selected from: —NH₂,        —N(H)R⁵, —N(R⁵)R⁶, (4-7C)heterocycloalkyl, optionally        substituted, one or more times, identically or differentially,        with a group selected from —NH₂, —N(H)R⁵, —N(R⁵)R⁶.    -   R⁵ and R⁶, independently from each other, represent a group        selected from a hydrogen atom and (1-3C)alkyl.    -   R⁷ represents a group selected from a hydrogen atom and        (1-3C)alkyl.    -   R⁸ represents a hydrogen atom

Another example of known TTK inhibitors are chemical compounds belongingto the class of pyrrolopyridines according to Formula VII orpharmaceutically acceptable salts thereof as described in WO2009/032694A1, WO2009/032703 A1 and Nature Chemical Biology 6 (2010), 359.

wherein,

-   -   R¹ is selected from the group consisting of (6-10C)aryl, wherein        said group optionally can be substituted;    -   R² is selected from the group consisting of (6-10C)aryl, wherein        said group optionally can be substituted.

Yet, another example of known TTK inhibitors are chemical compoundsbelonging to the class of aminoyridines and aminopyrimidines accordingto Formula VIII or pharmaceutically acceptable salts thereof asdescribed in WO2011/016472 A1, ACS Med. Chem. Letters 3 (2012), 560 andBioorg. Med. Chem. Letters 23 (2015), 2247.

wherein,

-   -   R¹ is selected from the group consisting of hydrogen atom or        amino;    -   R² is selected from the group consisting of (6-10C)aryl,        (1-5C)heteroaryl, (1-6C)alkyl, (3-6C)cycloalkyl and        (3-7C)heterocycloalkyl, wherein said groups optionally can be        substituted;    -   R³ is selected from the group consisting of (6-10C)aryl, wherein        said groups optionally can be substituted;    -   X is C or N.

The terms as used herein refer to the following:

Halogen means fluorine, chlorine, bromine or iodine.

-   (1-2C)Alkyl means an alkyl group having 1 to 2 carbon atoms, being    methyl or ethyl. A methyl group may be indicated as Me or CH₃.-   (1-3C)Alkyl means a branched or unbranched alkyl group having 1-3    carbon atoms, being methyl, ethyl, propyl or isopropyl.-   (1-4C)Alkyl means a branched or unbranched alkyl group having 1-4    carbon atoms, being methyl, ethyl, propyl, isopropyl, butyl,    isobutyl, sec-butyl or tert-butyl, (1-3C)alkyl groups being    preferred.-   (1-5C)Alkyl means a branched or unbranched alkyl group having 1-5    carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl,    isobutyl, sec-butyl, tert-butyl, pentyl and isopentyl, (1-4C)alkyl    groups being preferred.-   (1-6C)Alkyl means a branched or unbranched alkyl group having 1-6    carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl,    tert-butyl, n-pentyl and n-hexyl. (1-5C)alkyl groups are preferred,    (1-4C)alkyl being more preferred.-   (1-2C)Alkoxy means an alkoxy group having 1-2 carbon atoms, the    alkyl moiety having the same meaning as previously defined.-   (2-4C)Alkoxy means an alkoxy group having 2-4 carbon atoms, for    example ethoxy, propyloxy, butyloxy, isopropyloxy, isobutyloxy, and    tertbutyloxy. Ethyloxy and propyloxy being preferred. Ethyloxy    groups being more preferred.-   (1-3C)Alkoxy means an alkoxy group having 1-3 carbon atoms, the    alkyl moiety having the same meaning as previously defined.    (1-2C)Alkoxy groups are preferred.-   (1-4C)Alkoxy means an alkoxy group having 1-4 carbon atoms, the    alkyl moiety having the same meaning as previously defined.    (1-3C)alkoxy groups are preferred, (1-2C)alkoxy groups being most    preferred.-   (1-5C)Alkoxy means an alkoxy group having 1-5 carbon atoms, the    alkyl moiety having the same meaning as previously defined.    (1-4C)Alkoxy groups are preferred, (1-3C)alkoxy groups being more    preferred.-   (2-3C)Alkenyl means a branched or unbranched alkenyl group having    2-3 carbon atoms, such as ethenyl or 2-propenyl.-   (2-3C)Alkynyl means ethynyl or 2-propynyl.-   (3-4C)Cycloalkyl means a cycloalkyl group having 3-4 carbon atoms,    being cyclopropyl or cyclobutyl.-   (3-6C)cycloalkyl means a cycloalkyl group having 3-6 atoms. Examples    of “cycloalkyl” include, but are not limited to, cyclopropyl,    cyclobutyl, cyclopentyl or cyclohexyl.-   (2-5C)Heterocycloalkyl means a heterocycloalkyl group having 2-5    carbon atoms, preferably 3-5 carbon atoms; and one or two    heteroatoms selected from N, O and/or S, which may be attached via a    heteroatom if feasible, or a carbon atom. Preferred heteroatoms are    N or O. Preferred are oxetanyl, azetidinyl, piperidinyl,    morpholinyl, pyrrolidinyl and piperazinyl. Most preferred    (2-5C)heterocycloalkyl are oxetanyl and azetidinyl.-   (2-7C)Heterocycloalkyl means a heterocycloalkyl group having 2-7    carbon atoms, preferably 2-5 carbon atoms, and one or two    heteroatoms selected from N, O and/or S. Preferred heteroatoms are N    or O. Preferred (2-7C)heterocycloalkyl groups are azetidinyl,    pyrrolidinyl, piperidinyl, piperazinyl, homopiperidinyl, morpholinyl    or thiomorpholinyl. The heterocycloalkyl group may be attached via a    heteroatom if feasible.-   (6-10C)Aryl means an aromatic hydrocarbon group having 6-10 carbon    atoms. Examples of “(6-10C)aryl” include, but are not limited to,    phenyl, naphthyl, tetrahydronaphthyl or indenyl.-   (1-5C)Heteroaryl means a substituted or unsubstituted aromatic group    having 1-5 carbon atoms and 1-4 heteroatoms selected from N, O    and/or S. The (1-5C)heteroaryl may optionally be substituted.    Examples of “(1-5C)heteroaryl” include, but are not limited to,    tetrazolyl, imidazolyl, pyridyl, pyrimidyl, triazinyl, thienyl    furyl, pyrolyl or pyrazolyl.-   (3-6C)Cycloalkylamino means an amino group, monosubstituted with an    cycloalkyl group containing 3-6 carbon atoms having the same meaning    as previously defined.-   (1-6C)Alkylamino means an amino group, monosubstituted with an alkyl    group containing 1-6 carbon atoms having the same meaning as    previously defined. Preferred (1-6C)alkylamino group is methylamino.-   Di[(1-2C)alkyl]amino means an amino group, disubstituted with alkyl    group(s), each independently containing 1-2 carbon atoms and having    the same meaning as previously defined. Preferred    di[(1-2C)alkyl]amino group is dimethylamino.-   Di[(1-6C)alkyl]amino means an amino group, disubstituted with alkyl    group(s), each independently containing 1-6 carbon atoms and having    the same meaning as previously defined. Preferred    di[(1-6C)alkyl]amino group is N-methylpropan-1-amino.-   (2-7C)Heterocycloalkylamino means an amino group, monosubstituted    with a (2-7)heterocycloalkyl group containing 2-7 carbon atoms    having the same meaning as previously defined.-   (1-6C)Alkylaminocarbonyl means a carbonyl group substituted with an    amino group. Said amino group being monosubstituted with an alkyl    group having 1-6 carbon atoms and having the same meaning as    previously defined.-   (2-7C)Heterocycloalkylcarbonyl means a carbonyl group substituted    with an (2-7C)heterocycloalkyl group having 2-7 carbon atoms and    having the same meaning as previously defined.-   (1-5C)Alkoxycarbonyl means a carbonyl group substituted with an    alkoxy group the alkyl moiety of which having 1-6 carbon atoms as    previously defined.-   (1-6C)Alkylsulfonyl means a sulfonyl group substituted with an    (1-6C)alkyl group having 1-6 carbon atoms and having the same    meaning as previously defined.-   (1-6C)Alkylcarbonyl means a carbonyl group substituted with an    (1-6C)alkyl group having 1-6 carbon atoms and having the same    meaning as previously defined.-   (3-6C)Cycloalkylcarbonyl means a carbonyl group substituted with an    (3-6C)cycloalkyl group having 3-6 carbon atoms and having the same    meaning as previously defined.-   (1-6C)Alkylaminocarbonyl means a carbonyl group substituted with an    amino group. Said amino group being monosubstituted with an alkyl    group having 1-6 carbon atoms and having the same meaning as    previously defined.-   (1-6C)Alkylcarbonylamino means an amino group substituted with a    carbonyl group. Said carbonyl group being monosubstituted with an    alkyl group having 1-6 carbon atoms and having the same meaning as    previously defined.-   (3-6C)Cycloalkylcarbonylamino means an amino group substituted with    a carbonyl group. Said carbonyl group being monosubstituted with a    cycloalkyl group having 3-6 carbon atoms and having the same meaning    as previously defined.-   (2-7C)Heterocycloalkylcarbonylamino means an amino group substituted    with a carbonyl group. Said carbonyl group being monosubstituted    with a (2-7C)heterocycloalkyl group having 2-7 carbon atoms and    having the same meaning as previously defined.-   Hydroxy(1-2C)alkyl means a (1-2C)alkyl group having 1-2 carbon atoms    with the same meaning as previously defined, substituted with a    hydroxyl group.-   (1-2C)Alkoxy[(2-4C)alkoxy]_(n)(1-6C)alkyl means a (1-6C)alkyl group    having 1-6 carbon atoms with the same meaning as previously defined,    substituted with one or more (2-4C)alkyloxy groups, wherein n    represents an integer of 1, 2, 3 or 4, the alkoxy groups being    linearly connected one to another. The last (2-4C)alkyloxy group    being substituted with an (1-2C)alkyloxy group. In the    (1-2C)alkoxy[(2-4C)alkoxy]_(n)(1-6C)alkyl group, the preferred    (1-2C)alkoxy group is methoxy, the preferred (2-4C)alkoxy is ethoxy,    and the preferred (1-6C)alkyl is ethyl, preferably n is 1, 2, 3, 4,    n is 1 or 2 being most preferred.-   (1-9C)heteroaryl means a substituted or unsubstituted aromatic group    having 1-9 carbon atoms and 1-4 heteroatoms selected from N, O    and/or S. The (1-9C)heteroaryl may optionally be substituted.    Examples of “(1-9C)heteroaryl” include, but are not limited to,    quinolone, isoquinoline, indazole benzisoxazole and indole.-   (2-6C)alkenyl means a branched or unbranched alkenyl group having    2-6 carbon atoms. Examples of “(2-6C)alkenyl” include, but are not    limited to, ethenyl, 2-butenyl and n-pentenyl.-   (2-6C)alkynyl means a branched or unbranched alkynyl group having    2-6 carbon atoms, Examples of “(2-6C)alkynyl” include, but are not    limited to, ethynyl, propynyl, n-butynyl, n-pentynyl, isopentynyl,    isohexynyl or n-hexynyl.-   (3-7C)heterocycloalkyl means a heterocycloalkyl group having 3-7    carbon atoms, preferably 3-5 carbon atoms, and one or two    heteroatoms selected from N, O and/or S. Examples of    “heterocycloalkyl” include, but are not limited to, azetidinyl,    pyrrolidinyl, piperidinyl, homopiperidinyl or morpholinyl.-   (4-8C)heterocycloalkenyl) means a heterocycloalkenyl group having    4-8 carbon atoms, preferably 3-5 carbon atoms having a double bond    therein; and 1 heteroatom selected from N, O and/or S. Examples of    “heteroalkenyl” include, but are not limited to, oxycyclohexenyl and    azacyclohexenyl.-   Halo(1-6C)alkyl means a branched or unbranched alkyl group having    1-6 carbon atoms, in which from one up to all hydrogen atoms are    replaced by a halogen as defined herein. Examples of such branched    or straight chain haloalkyl groups useful in the present invention    include, but are not limited to, methyl, ethyl, propyl, isopropyl,    isobutyl and n-butyl substituted independently with one or more    halogen atoms, e.g., fluoro, chloro, bromo and iodo. Specific    examples of “haloalkyl include, but are not limited to,    fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl,    2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, and    perfluoro-n-propyl.-   HO(1-6C)alkyl means a branched or unbranched alkyl group having 1-6    carbon atoms, in which one, two or three hydrogen atoms are replaced    by a hydroxyl group. Examples of “HO(1-6C)alkyl” include, but are    not limited to, hydroxymethyl, 1-hydroxyethyl, 2-hydroxyethyl, and    1,2-dihydroxyethyl.-   H₂N(1-6C)alkyl means a branched or unbranched alkyl group having 1-6    carbon atoms, in which one, two or three hydrogen atoms are replaced    by an amino group. Examples of “H₂N(1-6C)alkyl” include, but are not    limited to, aminomethyl, 1-aminoethyl, 2-aminoethyl, and    1,2-di-aminoethyl.-   Cyano(1-6C)alkyl means a branched or unbranched alkyl group having    1-6 carbon atoms, in which one, two or three hydrogen atoms are    replaced by a cyano group. Examples of “cyano(1-6C)alkyl” include,    but are not limited to, cyanomethyl, 1-cyanoethyl, 2-cyanoethyl, and    1,2-dicyanoethyl.

In the above definitions with multifunctional groups, the attachmentpoint is at the last group.

When, in the definition of a substituent, is indicated that “all of thealkyl groups” of said substituent are optionally substituted, this alsoincludes the alkyl moiety of an alkoxy group.

The term “substituted” means that one or more hydrogens on thedesignated atom/atoms is/are replaced with a selection from theindicated group, provided that the designated atom's normal valencyunder the existing circumstances is not exceeded, and that thesubstitution results in a stable compound. Combinations of substituentsand/or variables are permissible only if such combinations result instable compounds.

“Stable compound” or “stable structure” is defined as a compound orstructure that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture, and formulation into anefficacious therapeutic agent.

The term “optionally substituted” means optional substitution with thespecified groups, radicals or moieties.

The present invention will now be described in the following examples.These examples are intended to be illustrative of the invention, and arenot intended to be limiting of the invention.

EXAMPLES Methods Cancer Cell Lines

To determine whether sensitivity of cancer-derived cells to TTKinhibitors correlates with the presence of a specific genomic marker,various TTK inhibitors were profiled in parallel on a panel of sixty-sixcancer cell lines derived from different tumor origins and that havebeen characterized with respect to the expression and mutant status ofvarious oncogenes and tumor suppressor genes (Uitdehaag, J. C. M., etal., PLoS ONE 9(3), e92146; 2014). The cancer cell lines used are listedin Table 1. All cell lines were purchased from the American Type CultureCollection (ATCC) (Manassas, Va., U.S.A.).

TABLE 1 Cancer cell lines used in drug sensitivity analysis. 769-P AN3CA Daoy KU812 OVCAR-3 SUP-T1 786-0 AsPC-1 DLD-1 LNCaP FGC PA-1 SW48A-172 AU-565 DoTc2 4510 LoVo RKO SW480 A-204 BT-20 DU145 LS 174TRPMI-7951 SW620 A375 BT-549 FaDu MCF7 RT4 SW948 A388 BxPC-3 HCT116 MeWoSHP-77 T24 A-427 C-33A HCT-15 MG-63 SJRH30 T986 A-498 CAL27 HS 578T MIAPaCa-2 SK-N-AS TT A-549 CCRF-CEM J82 MOLT-4 SK-N-FI U-2 OS A-707 COLO205Jurkat E6-1 NCI-H460 SNU-C2B U-87 MG ACHN COLO829 K-562 NCI-H82 SRWA-ES-BJ

The genetic status of the thirty-one most frequently changed cancergenes in the cell line panel has been established as either ‘mutant’ or‘wild-type’ from public sequencing data (Garnett, M. J., et al., Nature483: 570; 2012). In Table 2 the cell lines are listed that have CTNNB1gene mutations. A427, LS 174T, HCT116 and SW48 have mutations in theserine or threonine residues that regulates the stability of β-cateninvia phosphorylation at specific serine and threonine residues (Polakis,P., Curr. Opin. Gen. Dev. 9: 15; 1999). The other cell lines listed inthe table and the cell lines from the sixty-six cancer cell line panelthat are not mentioned, either have CTNNB1 mutations that are notimplicated in regulation of protein stability, or do not have any CTNNB1gene mutation.

TABLE 2 CTNNB1 gene mutations in cancer cell lines included in the drugsensitivity analysis. Amino Mutations in acid Literature regulatory Cellline Codon change change Zygosity reference domain A427 121A > G T41Ahomozygous 1,2 Yes LS 174T 134C > T S45F 2,3 Yes HCT 116 131-133delCTTS45del heterozygous 1,2,4,5 Yes SW48 98C > A S33Y heterozygous 1,2,4,5Yes Other C33A 1216G > A P406I heterozygous 1 no DU145 914C > G A305Ghomozygous 1,4 no LNCap 1154T > A I385H heterozygous 1 no T98G 526C > TB176Y heterozygous 1 no U2OS 1584-1G > A heterozygous 1 no (intronicsubstitution) COLO205 E562 splice diploid 4 no References 1. Cosmic CellLines project, status Feb. 2^(nd), 2015 2. Garnett, M. J., et al. 3.Wang, Z., et al., Cancer Res. 63: 5234; 2003 4. Cancer Cell LineEncyclopedia, status Feb. 2^(nd), 2015 5. Morin, et al., 1997; Ilyas,M., et al., Proc. Natl. Acad. Sci. USA 94: 10330; 1997

Cell Proliferation Assays

All cell lines were cultured in the media as recommended by ATCC. Theculture media were purchased from Life Technologies (Bleiswijk, TheNetherlands). Proliferation assays were carried out as described(Uitdehaag J. C. M., et al.) in 384-well plates with incubation withcompound for 120 hours. Effects of TTK inhibitors were measured in a9-point dilution series in duplicate. The final DMSO concentrationduring incubation was 0.4% (v/v) in all wells. As readout, intracellularATP content was used as an indirect measure of cell number, usingATPlite™ 1 Step solution (Perkin Elmer, Groningen, The Netherlands). Theeffect of the compounds on cell growth was calculated relative tocontrol wells containing only 0.4% (v/v) DMSO. Half maximum inhibitorypotencies (IC₅₀s) were fitted by non-linear regression using XLfit™ 5(ID Business Solutions, Ltd., Surrey, U.K.).

Analysis of Cell Panel Response Data

Analysis of variance (Anova) was used to determine whether there was astatistical correlation between a particular genetic change in the panelof cell lines and drug sensitivity. The mutations and the ¹⁰ log IC₅₀from the cell proliferation assays were analyzed with a type II Anovaanalysis using the statistical program R (R Foundation for statisticalcomputing, Vienna, Austria) and displayed in volcano plots such as shownin FIG. 2. The p-value (y-axis in the volcano plot) indicates theconfidence level for genetic association of mutations in a particulargene with a IC₅₀ shift. The average factor with which the IC₅₀ shifts isindicated on the x-axis. The areas of the circles are proportional tothe number of mutants in the cell panel (each mutation is present atleast twice). To compute significance, p-values were subjected to aBenjamini-Hochberg multiple testing correction (Benjamini, Y., andHochberg, Y., J. Royal. Statistic. Soc. B 57:289; 1995). Geneticassociations with a<20% false discovery rate were consideredsignificant.

Statistical Analysis of Difference in Sensitivity

To quantify differences in sensitivity between CTNNB1-mutant and CTNNB1proficient, the inhibitory potency of the TTK inhibitors was expressedas pIC₅₀ (−¹⁰ log IC₅₀). A two-tailed Student's t-test was performed todetermine whether differences in sensitivity (ΔpIC₅₀) between CTNNB1mutant and CTNNB1 proficient cells were statistically significant (i.e.,p<0.05).

Comparison of Sensitivity in Isogenic Cell Lines

To determine whether mutated CTNNB1 was sufficient to confer increasedsensitivity to TTK inhibitors, proliferation assay were performed with apair of isogenic cell lines. Parental HCT116 cells harbor a deletion ofthree base pairs in one copy of the CTNNB1 gene, resulting in deletionof the regulatory serine residue at position 45 (S45del) of β-catenin(Table 2). Parental HCT116 cells are furthermore heterozygous regardingmutation in the CTNNB1 gene, i.e., the genotype of parental HCT116regarding CTNNB1 is S45del/+. An isogenic cell line derived from HCT116lacking the mutated CTNNB1 gene copy (+/−) was purchased from HorizonDiscovery (Cambridge, U.K.) (Chan, T. A., et al., Proc. Natl. Acad. Sci.USA 99: 8265; 2002). HCT116 parental and isogenic derivatives werecultured in identical media, as recommended by the supplier.Proliferation assays were carried out as described for cancer cell lines(Uitdehaag J. C. M., et al.). Dose response curves were plotted, IC₅₀,pIC₅₀ and maximum percentage effect (efficacy) were calculated usingXLfit™ 5. Difference in sensitivity of the parental and the isogenicderivative were expressed as difference in pIC₅₀ (ΔpIC₅₀) and differencein efficacy (Δefficacy).

TTK Inhibitors (Examples 1 to 31)

The following examples are illustrative embodiments of the invention,not limiting the scope of the invention in any way. Reagents are eithercommercially available or are prepared according to procedures in theliterature.

Method LCMS (A)

Method name NTRC_C18_Short.M Column Waters XTerra C18-MS, 50 × 4.6 mmID, 2.5 μm Flow 0.5 ml/min. Temperature 40° C. Detector DAD 210, 254,280 nm Detector MSD API-ES MSD signal 1 2 Mode Scan Scan PolarityPositive Negative Mass Range 100-1000 m/z 100-1000 m/z Fragmentor 70 70Cycle Time 50% 50% Sample N/A preparation Concentration 1 mg/ml in MeOHor CAN Injection volume 1.0 μl Eluent A B Time % 0.1% Formic % 0.05%Formic Acid in [min] Acid Acetonitrile 0 90 10 0.3 90 10 7.0 10 90 7.190 10 10.0 90 10 Post time 0.2 min Stop time 10 min

Method LCMS (B)

Method name NTRC_C18.M Column Waters XTerra C18-MS, 50 × 4.6 mm ID, 2.5μm Flow 0.5 ml/min. Temperature 40° C. Detector DAD 210, 254, 280 nmDetector MSD API-ES MSD signal 1 2 Mode Scan Scan Polarity PositiveNegative Mass Range 100-1000 m/z 100-1000 m/z Fragmentor 70 70 CycleTime 50% 50% Sample N/A preparation Concentration 1 mg/ml in MeOH or CANInjection volume 1.0 μl Eluent A B Time % 0.1% % 0.05% Formic Acid in[min] Formic Acid Acetonitrile 0 90 10 1 90 10 22.0 10 90 22.1 90 1030.0 90 10 Post time 0.2 min Stop time 30 min

Method LCMS (C)

LC System HP1200SL Column Agilent Eclipse plus C18 150 mm × 2.1 mm ID3.5 μm Column temperature 40° C. Sample(s) ca 1 mg/mL Autosampler 20° C.temperature Injection volume 5 μl Flow 0.5 ml/min Type of Pump BinaryEluent A = MilliQ + 0.1% Formic Acid B = Acetonitrile time (min) % A % BGradient 0 90 10 1 90 10 22 10 90 22.1 90 10 30 90 10 Next Injectiondelay 0 min UV detection UV 210, 240, 280 nm Flowcell DAD 10 mm

MS system Agilent 6130 single Quad MS Source ESI Mode Positive (+) Massrange 100-1000 Da Flow The total flow was split to a suitable flowinfused directly in the APCI/ESI multimode source of the Agilent 6130

Method Preparative HPLC

LC System Waters Prep System Column Phenomenex Luna, C18(2) 100 A, 150mm × 21.2 mm, 5 μm Column Temp 20° C. Sample(s) 10-50 mg Autosamp. Temp20° C. Injection volume 500-950 μL Flow 15 ml/min Eluent A = MilliQ +MeCN (9/1) B = Acetonitrile time (min) % A % B % C Gradient 0 97 0 3 2037 60 3 25 37 60 3 25.1 97 0 3 30 97 0 3 UV detection Photo Diode Array

The following abbreviations are used throughout the application withrespect to chemical terminology:

-   TFA Trifluoracetic acid-   HATU    O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate-   DMF N,N-Dimethylformamide-   THF Tetrahydrofuran-   MeOH Methanol-   EtOAc Ethyl acetate-   DCM Dichloromethane-   Na₂SO₄ Sodium sulfate-   TMS-Cl Chlorotrimethylsilane-   DiPEA N,N-Diisopropylethylamine-   EtOH Ethanol-   10% Pd/C 10% Palladium on charcoal-   HPLC High Performance Liquid Chromatography-   LCMS Liquid Chromatography with Mass Spectrometry detection-   NaOH Sodium hydroxide-   KOH Potassium hydroxide-   HCl Hydrogen chloride-   NaHCO₃ Sodium bicarbonate-   4-DMAP 4-Dimethylamino pyridine-   Boc tert-Butyloxycarbonyl-   Cbz Benzyloxycarbonyl-   HNO₃ Nitric acid-   LiHMDS Lithium bis(trimethylsilyl)amide-   DDQ 2,3-Dichloro-5,6-dicyano-p-benzoquinone-   DEAD Diethyl azodicarboxylate-   o/n overnight

The names of the final products in the examples are generated usingAccelrys Draw (version 4.1).

Example 1 (WITJ0018D)

N6-cyclohexyl-N2-(2-methyl-4-morpholino-phenyl)-9H-purine-2,6-diamine

This compound was prepared as described in WO2010/111406 A2 and Bioorg.Med. Chem. Letters 22 (2012) 4377. Purification was performed usingpreparative HPLC to afford the title compound (338 mg). Data: LCMS (C)R_(t): 10.995 min; m/z 408.3 (M+H)⁺.

Example 2 JGS0282C)

N-cyclopropyl-4-[8-(isobutylamino)imidazo[1,2-a]pyrazin-3-yl]benzamide

This compound was prepared as described in WO2012/080229 A1 and CellDeath and Differentiation 20 (2013), 1532. Purification was performedusing preparative HPLC to afford the title compound (47 mg). Data: LCMS(B) R_(t): 8.088 min; m/z 350.2 (M+H)⁺.

Example 3 (BTHO238B)

N-(2,6-diethylphenyl)-1-methyl-8-[4-[(1-methyl-4-piperidyl)carbamoyl]-2-(trifluoromethoxy)anilino]-4,5-dihydropyrazolo[4,3-h]quinazoline-3-carboxamide

This compound was prepared as described in WO2009/156315 A1 and CancerRes. 70 (2010), 10255. Purification was performed using preparative HPLCto afford the title compound (191 mg). Data: LCMS (A) R_(t): 5.810 min;m/z 677.6 (M+H)⁺.

Example 4 (WITJ113B)

N-(2,6-diethylphenyl)-8-(2-methoxy-4-piperazin-1-yl-anilino)-1-methyl-4,5-dihydropyrazolo[4,3-h]quinazoline-3-carboxamide

This compound was prepared as described in WO2009/156315 A1.Purification was performed using preparative HPLC to afford the titlecompound (7.3 mg). Data: LCMS (C) R_(t): 12.954 min; m/z 567.3 (M+H)⁺.

Example 5 (JGS0716D)

N-cyclopropyl-4-[6-(2,3-difluoro-4-methoxy-phenoxy)-8-(tetrahydropyran-4-ylmethylamino)imidazo[1,2-b]pyridazin-3-yl]-2-methyl-benzamide

This compound was prepared as described in WO 2014/131739 A1.Purification was performed using preparative HPLC to afford the titlecompound (90 mg). Data: LCMS (B) R_(t): 13.496 min; m/z 564.5 (M+H)⁺.

Example 6

N-cyclopropyl-4-[6-(3-fluoro-4-methoxy-phenoxy)-8-(oxetan-3-ylmethylamino)imidazo[1,2-b]pyridazin-3-yl]-2-methyl-benzamide

This compound was prepared as described in WO 2014/131739 A1.Purification was performed using preparative HPLC to afford the titlecompound (45 mg). Data: LCMS (B) R_(t): 11.640 min; m/z 518.4 (M+H)⁺.

Intermediate 1

Ethyl 2-chloro-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate (a)5-Bromo-2-chloro-pyrimidin-4-amine (WITJ0221)

To a solution of 5-bromo-2,4-dichloro-pyrimidine (150 g; 658 mmol) inTHF (445 mL) was added ammonium hydroxide (25% in water, 250 mL) and theresulting reaction mixture was stirred at room temperature for 90 min.The mixture was subsequently concentrated in vacuo to a small volume andpartitioned between ethyl acetate and water. The organic phase wasseparated and washed with water and brine, dried over sodium sulfate,filtered and concentrated to give 137.3 g (quant. yield) of5-bromo-2-chloro-pyrimidin-4-amine.

(b) 5-Bromo-2-methoxy-pyrimidin-4-amine (WITJ0223)

To a suspension of 5-bromo-2-chloro-pyrimidin-4-amine (137.3 g, 658mmol) in methanol (1 L) was added portion-wise sodium methoxide (83.5 g;1.54 mol). The reaction mixture was stirred for 2 h. at reflux. Thereaction mixture was concentrated to a small volume (˜400 mL) and pouredinto a saturated solution of ammonium chloride in water (1.2 L). Thismixture was allowed to stir for 15 min, after which the water layer wasextracted with ethyl acetate. The combined ethyl acetate layers werewashed with brine, dried over sodium sulfate, filtered and concentratedto yield 5-bromo-2-methoxypyrimidin-4-amine (133.7 g, 99.4%).

(c) Ethyl (E)-3-(4-amino-2-methoxy-pyrimidin-5-yl)prop-2-enoate 256)(WITJ0256)

Palladium(II) acetate (1.21 g, 5.5 mmol) and triphenylphosphine (3.40 g,13.0 mmol) were dissolved in anhydrous and oxygen-free DMF (53 mL) andstirred for 5 min at 30° C. to give an orange suspension. To thissuspension was added a solution of 5-bromo-2-methoxypyrimidin-4-amine(44.1 g, 216 mmol) in DMF (270 mL), triethylamine (60.2 mL, 432 mmol)and a solution of ethyl acrylate (23.5 mL, 216 mmol) in DMF (50 mL). Thereaction mixture was stirred at 100° C. o/n under a nitrogen atmosphere.The reaction mixture was evaporated to a small volume. Water (300 mL)and brine (300 mL) were added to the mixture, followed by an extractionwith ethyl acetate (300 mL, twice). The combined organic layers werewashed with water, brine, dried over sodium sulfate and concentrated invacuo. The crude product was purified by silica column chromatography(ethyl acetate:heptane=2:1 v/v %) to yield the title compound (38.2 g,77%).

(d) 2-Methoxy-6,8-dihydro-5H-pyrido[2,3-d]pyrimidin-7-one (WITJ0262)

To a stirred solution of ethyl(E)-3-(4-amino-2-methoxy-pyrimidin-5-yl)prop-2-enoate (12.52 g, 56.1mmol) in methanol (250 mL) was added a suspension of 10% Pd on charcoal(1.19 g) in methanol/ethanol=3/1 v/v % (30 mL). The reaction mixture wasstirred at room temperature for 15 min under nitrogen atmosphere. Then,ammonium formate (35.3 g, 561 mmol) was added and the resulting reactionmixture was refluxed o/n. After cooling of the reaction mixture, a freshportion of ammonium formate (20 g, 317 mmol) was added and stirring wascontinued an additional night at reflux. The reaction mixture wasfiltered over Decalite® and the Pd—C/Decalite® residue was washed withdichloromethane/methanol=8/2 v/v % and the filtrate was concentrated invacuo. The residue was dissolved in dichloromethane and washed withwater, dried over sodium sulfate, filtered and concentrated in vacuo toobtain 9.4 g (94%) of2-methoxy-6,8-dihydro-5H-pyrido[2,3-d]pyrimidin-7-one.

(e) Ethyl2-methoxy-5,6,8,9-tetrahydropyrimido[4,5-e]indolizine-7-carboxylate(JGS0241)

2-Methoxy-6,8-dihydro-5H-pyrido[2,3-d]pyrimidin-7-one (4.79 g, 26.8mmol) was suspended in THF (200 mL) in a three-necked flask (500 mL),equipped with a mechanical stirrer, a thermometer and a refluxcondenser. The mixture was cooled to 0° C. and sodium hydride (60%dispersion in oil, 1.18 g, 29.4 mmol) was added in two batches. Themixture was stirred at 0° C. for 30 min.(1-ethoxycarbonylcyclopropyl)triphenylphosphonium tetrafluoroborate(13.6 g, 29.4 mmol) was added and the resulting suspension was heated toreflux and kept at reflux temperature for 3 days. The reaction mixturewas cooled to room temperature and poured in a 1/1/1 mixture ofbrine/water/EtOAc (450 mL). The water layer was extracted with ethylacetate (2×). The combined organic layers were washed with water andbrine, dried over sodium sulfate, filtered and concentrated in vacuo togive 18.05 g of an orange oil. The crude product was used directly inthe next step without purification.

(f) Ethyl 2-methoxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(JGS244)

To a stirred solution of ethyl2-methoxy-5,6,8,9-tetrahydropyrimido[4,5-e]indolizine-7-carboxylate(18.05 g, 26.2 mmol) in dichloromethane (100 mL) was added acetic acid(3.15 g, 3 mL) and lead(IV)acetate (13.9 g, 31.4 mmol). The reactionmixture was stirred for 2 h at room temperature then filtered over a PEfilter to remove Pb-salts and the Pb-residue was washed with 2×30 mLDCM. The filtrate was concentrated in vacuo and the resulting residuewas dissolved in ethyl acetate (300 mL). A solution of sodiumbicarbonate (5%) was added until pH˜8.5. Both the organic and the waterlayers were filtered over Decalite® to remove any remaining salts. Thewater layer was subsequently extracted with EtOAc (2×50 mL). Thecombined organic layers were washed with 5% sodium bicarbonate-solution(100 mL), water (100 mL), brine (50 mL), dried (Na₂SO₄), filtered andconcentrated in vacuo. The crude product was purified by columnchromatography on silica (heptane: ethyl acetate=I/O to 1/1 v/v %) toyield the title compound (4.74 g, 66% over two steps).

(g) Ethyl 2-hydroxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(JGS0245)

Sodium iodide (7.83 g, 52.2 mmol) was added to a stirred solution ofethyl 2-methoxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate (4.74g, 17.3 mmol) in acetonitrile (150 mL). Trimethylsilyl chloride (5.64 g,6.59 mL) dissolved in acetonitrile (30 mL) was added drop-wise to thereaction mixture and the mixture was stirred at room temperature o/n.NaI (1 eq) was added and additional TMS-Cl (0.94 g, 1.1 mmol) inacetonitrile (6 mL) was added drop-wise and the reaction was stirred for3 days at room temperature. The mixture was concentrated and the residuewas suspended in 200 mL DCM/MeOH (4/1) and extracted with a mixture ofsaturated solution of sodium thiosulfate (200 mL) and water (200 mL).The water layer was extracted with 3×150 mL DCM/MeOH (4/1). The combinedorganic layers were dried over sodium sulfate, filtered and the solventwas removed under reduced pressure to give a yellow solid. The residuewas dried at 40° C. under vacuum for 18h to give 3.89 g ethyl2-hydroxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate (86%).

(h) Ethyl 2-chloro-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(Intermediate 1) (JGS0278)

N,N-dimethylaniline (182 mg, 191 uL, 1.50 mmol) was added to a solutionof ethyl 2-hydroxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(3.89 g, 15.0 mmol) in acetonitrile (100 mL). A solution ofphosphorus(V) oxychloride (11.5 g, 7.00 mL, 75.0 mmol) in acetonitrile(15 mL) was added drop-wise to the reaction mixture. The yellowsuspension was heated for 4 hours to 65° C. during which the suspensionturned into a clear solution. After cooling, the mixture was slowlypoured in a stirred mixture of 25% aq. ammonia (200 mL, 86.7 eq.) andice-water (250 mL) keeping the temperature below 10° C. in 15-20minutes. After stirring for another 15 minutes the solids were filtered.The solids were dissolved in 200 mL EtOAc and washed with brine (20 mL).The organic layer was dried over sodium sulfate, and concentrated invacuo to give an off-white solid. The crude product was purified bycolumn chromatography on silica (heptane/ethyl acetate=1/0 to 1/1 v/v %)to yield the title compound (3.05 g, 73%).

Intermediate A

Benzyl 4-(4-amino-3-methyl-phenyl) piperazine-1-carboxylate (a) Benzyl4-(3-methyl-4-nitro-phenyl)piperazine-1-carboxylate (WITJ404)

Benzyl piperazine-1-carboxylate (1.05 mL, 5.25 mmol) and potassiumcarbonate (1.38 g, 10 mmol) were added to a solution of4-fluoro-2-methyl-1-nitro-benzene (776 mg, 5 mmol) in DMF (10 mL) andthe resulting mixture was stirred at 100° C. for 18 h. Water was addedto the reaction mixture and extraction performed with ethyl acetate. Thecombined organic layers were washed with brine, dried over sodiumsulfate and concentrated in vacuo. The crude product was purified bysilica column chromatography (heptane/ethyl acetate=I/O to 6/4 v/v %) toyield the title compound (1.75 g, 98%).

(b) Benzyl 4-(4-amino-3-methyl-phenyl) piperazine-1-carboxylate(Intermediate A) (WITJ406)

Benzyl 4-(3-methyl-4-nitro-phenyl)piperazine-1-carboxylate (355 mg, 1mmol) was dissolved in THF (5 mL) and acetic acid (1.1 mL) was added.The mixture was cooled to 0° C. and zinc (1.31 g, 20 mmol) was added insmall portions to keep the temperature below 20° C. The reaction mixturewas stirred at room temperature o/n. After TLC analysis indicated acomplete conversion of the starting material, the mixture was filteredover Decalite® and the Zn-Decalite® residue was washed with EtOAc (20mL). The combined filtrates were washed with a 1N NaOH-solution (25 mL),followed by water (25 mL) and brine (25 mL). The organic layer was dried(Na₂SO₄), filtered and concentrated in vacuo to give benzyl4-(4-amino-3-methyl-phenyl)piperazine-1-carboxylate (327 mg,quantitative).

Intermediate 2

Benzyl4-[4-[(7-chlorocarbonyl-5,6-dihydropyrimido[4,5-e]indolizin-2-yl)amino]-3-methyl-phenyl]piperazine-1-carboxylate(a) Ethyl2-[4-(4-benzyloxycarbonylpiperazin-1-yl)-2-methyl-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(WITJ407)

To a suspension of ethyl2-chloro-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(Intermediate 1, 292 mg, 1.05 mmol) in n-butanol (8 mL) was added benzyl4-(4-amino-3-methyl-phenyl)piperazine-1-carboxylate (Intermediate A, 327mg, 1.0 mmol) and trifluoroacetic acid (153 L 2.0 mmol). The reactionmixture was heated for 12 hours at 120° C. under microwave radiation.The reaction mixture was concentrated in vacuo and the residue wasdissolved in ethyl acetate. The organic layer was washed with asaturated solution of sodium bicarbonate, dried over sodium sulfate,filtered and concentrated in vacuo. The crude product was purified bysilica column chromatography (heptane/ethyl acetate=4/6 to 0/1 v/v %).Fractions containing product were collected and evaporated to affordethyl2-[4-(4-benzyloxycarbonylpiperazin-1-yl)-2-methyl-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(423 mg, 75% yield).

(b) Benzyl4-[4-[(7-chlorocarbonyl-5,6-dihydropyrimido[4,5-e]indolizin-2-yl)amino]-3-methyl-phenyl]piperazine-1-carboxylate(Intermediate 2) (WITJ408/WITJ414)

To a solution of ethyl2-[4-(4-benzyloxycarbonylpiperazin-1-yl)-2-methyl-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(423 mg, 0.75 mmol) in 15 mL absolute ethanol was added a 2MNaOH-solution (935 μL (2.5 eq). 1.87 mmol). The reaction mixture washeated at 65° C. o/n. Reaction mixture was evaporated to dryness anddried under high vacuum. The resulting residue was dissolved in water,stirred o/n at room temperature and lyophilised to yield the crudesodium2-[4-(4-benzyloxycarbonylpiperazin-1-yl)-2-methyl-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate.

Thionyl chloride (561 μL, 7. mmol) was added to a cold (0° C.)suspension of the crude sodium2-[4-(4-benzyloxycarbonylpiperazin-1-yl)-2-methyl-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(217 mg, 0.39 mmol theor.) in dichloromethane (8 mL). The resultingslurry was stirred at room temperature o/n. The reaction mixture wasconcentrated in vacuo and the residue was co-evaporated with toluene(2×10 mL) to give of benzyl4-[4-[(7-chlorocarbonyl-5,6-dihydropyrimido[4,5-e]indolizin-2-yl)amino]-3-methyl-phenyl]piperazine-1-carboxylateas a yellow/brown powder (261 mg, quant. crude yield).

Example 7 (WITJ0416/WITJ429A)

N-(2,6-dimethylphenyl)-2-(2-methyl-4-piperazin-1-yl-anilino)-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

To a suspension of benzyl4-[4-[(7-chlorocarbonyl-5,6-dihydropyrimido[4,5-e]indolizin-2-yl)amino]-3-methyl-phenyl]piperazine-1-carboxylate(Intermediate 2, 45 mg, 0.081 mmol theor.) in acetonitrile (3 mL) wasadded 2,6-dimethylaniline (15 μL, 0.12 mmol) and a catalytic amount of4-DMAP. The reaction mixture was stirred at 50° C. for 1 h. Afterevaporation of the solvent, the Cbz-group was de-protected usingTFA/thioanisole and the crude product was purified by preparative HPLC.Fractions containing product were collected and concentrated in vacuo.The residue was partitioned between dichloromethane and 5%NaHCO₃-solution. The organic phase was separated over a PE-filter andevaporated to afford the title compound (20 mg, 64%). Data: LCMS (B)R_(t): 9.706 min; m/z 508.3 (M+H)⁺.

Example 8 (JGS439C)

N-(2,6-dimethylphenyl)-2-[2-methoxy-4-(tetrahydropyran-4-ylcarbamoyl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide(a)2-(4-Bromo-2-methoxy-anilino)-N-(2,6-dimethylphenyl)-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide(JGS453)

This compound was prepared from its corresponding acid chloride, usingthe same sequence of reactions, as described for Intermediate 2, usingcommercially available 4-bromo-2-methoxyaniline as starting material.The acid chloride was subsequently reacted with 2,6-dimethylanilineaccording to procedures described in Example 7 to afford the titlecompound (1.35 g, 84%).

(b)2-(4-Cyano-2-methoxy-anilino)-N-(2,6-dimethylphenyl)-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide(JGS455)

To a solution of2-(4-bromo-2-methoxy-anilino)-N-(2,6-dimethylphenyl)-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide(1.35 g, 2.6 mmol) and zinc cyanide (321 mg, 2.73 mmol) in DMF (4 mL)was added tetrakis(triphenylphosphine)palladium(0) (300 mg, 0.26 mmol).The reaction mixture was heated for 30 minutes at 170° C. undermicrowave radiation. After cooling to ambient temperature, the mixturewas concentrated and the residue was diluted with ethyl acetate, washedwith water and brine, dried over sodium sulfate, filtered andconcentrated in vacuo to afford the crude title compound (1.05 g, 87%).

(c)4-[[7-[(2,6-Dimethylphenyl)carbamoyl]-5,6-dihydropyrimido[4,5-e]indolizin-2-yl]amino]-3-methoxy-benzoicacid (JGS0457)

To a stirred suspension of2-(4-cyano-2-methoxy-anilino)-N-(2,6-dimethylphenyl)-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide(750 mg, 1.61 mmol) in MeOH (25 mL) was added a solution of potassiumhydroxide (453 mg, 8.07 mmol) in water (12.5 mL). The reaction mixturewas heated for 2 hours at 120° C. under microwave radiation. Afterevaporation of the methanol fraction, the resulting water layer wasacidified by addition of 2N HCl-solution until pH-2. After extractionwith dichloromethane, the combined organic layers were filtered over aPE-filter to give 330 mg of the title compound (yield: 42%).

(d)N-(2,6-dimethylphenyl)-2-[2-methoxy-4-(tetrahydropyran-4-ylcarbamoyl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide(JGS439C)

4-[[7-[(2,6-Dimethylphenyl)carbamoyl]-5,6-dihydropyrimido[4,5-e]indolizin-2-yl]amino]-3-methoxy-benzoicacid (30 mg, 0.062 mmol) was dissolved in N,N-dimethylformamide (3 ml).HATU (25.9 mg, 0.068 mmol) and N,N-diisopropylethylamine (43.1 μL, 0.25mmol) were subsequently added and the mixture was stirred for 10 min atroom temperature. 4-Aminotetrahydropyran hydrochloride (12.8 mg, 0.093mmol) was added and the mixture was stirred at room temperature o/n. Themixture was poured into a mixture ethyl acetate/water/brine (1/1/1) andstirred for 15 min. The organic layer was separated, washed with brine,dried over sodium sulphate filtered and concentrated in vacuo.Purification was performed using preparative HPLC to afford the titlecompound (5 mg, 18%). Data: LCMS (B) R_(t): 14.407 min; m/z 567.3(M+H)⁺.

Intermediate B

2-Methoxy-4-(1,3,5-trimethylpyrazol-4-yl)aniline (a) tert-ButylN-[2-methoxy-4-(1,3,5-trimethylpyrazol-4-yl)phenyl]carbamate (NV0068)

A mixture of tert-butyl N-(4-bromo-2-methoxy-phenyl)carbamate (150 mg,0.5 mmol),1,3,5-trimethyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)pyrazole(118 mg, 0.5 mmol), tetrakis(tri-phenylphosphine)palladium(0) (58 mg,0.05 mmol) and potassium carbonate (207 mg, 1.5 mmol) in dioxane (4 mL)was heated at 100° C. under microwave irradiation for 20 minutes in asealed tube. After cooling to ambient temperature, the mixture wasconcentrated and the residue was diluted with ethyl acetate, washed withwater and brine, dried over sodium sulfate, filtered and concentrated invacuo. The residue was purified by column chromatography (heptane/ethylacetate=100/0 to 25/75 v/v %) to afford tert-butylN-[2-methoxy-4-(1,3,5-trimethylpyrazol-4-yl)phenyl]carbamate (126.8 mg,77%).

(b) 2-Methoxy-4-(1,3,5-trimethylpyrazol-4-yl)aniline (Intermediate B)(NV0076)

tert-Butyl N-[2-methoxy-4-(1,3,5-trimethylpyrazol-4-yl)phenyl]carbamate(127 mg, 0.38 mmol) was dissolved in DCM (2 mL). TFA (3 mL) was addedand the reaction mixture was stirred for 1 hour at room temperature. Themixture was concentrated in vacuo to give a brown oil (313 mg) that wasused without further purification.

Intermediate C (JDM0438/JDM035)

1-[2-[2-(2-Methoxyethoxy)ethoxy]ethyl]-3,5-dimethyl-pyrazol-4-amine (a)1-[2-[2-(2-Methoxyethoxy)ethoxy]ethyl]-3,5-dimethyl-4-nitro-pyrazole

To a cold (0° C.) solution of 3,5-dimethyl-4-nitro-1H-pyrazole (250 mg,1.77 mmol), triethylene glycol monomethylether (482 μL, 3.01 mmol) andtriphenylphosphine (789 mg, 3.01 mmol) in THF (10 mL) was added dropwisea solution of 40% DEAD in toluene (1.31 mL, 3.01 mmol) The reactionmixture was allowed to warm to room temperature and was stirred for 3 h.Ethyl acetate was added and washed with a 10% NaCl-solution. The organiclayer was dried (Na₂SO₄), filtered and concentrated. The residue waspurified by column chromatography (DCM/MeOH=99/1 to 95/5 v/v %) toafford1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]-3,5-dimethyl-4-nitro-pyrazole(1.7 g, crude) which was used without purification in the next step.

(b) 1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]-3,5-dimethyl-pyrazol-4-amine(Intermediate C)

1-[2-[2-(2-Methoxyethoxy)ethoxy]ethyl]-3,5-dimethyl-4-nitro-pyrazole(1.5 g, 1.77 mmol theor.) was dissolved in THF (15 mL) and acetic acid(1.6 mL) was added. The mixture was cooled to 0° C. and zinc (2.3 g,35.4 mmol) was added in small portions keeping the temperature below 20°C. The reaction mixture was stirred at room temperature o/n. After TLCanalysis indicated a complete conversion of the starting material, themixture was filtered over Decalite® and the Zn-Decalite® residue waswashed with ethyl acetate. The combined filtrates were washed with a 1NNaOH-solution, followed by water and brine. The organic layer was dried(Na₂SO₄), filtered and concentrated in vacuo. The residue was dissolvedin methanol and then filtered over a SCX-2 column. After rinsing thecolumn with methanol, the desired product was eluted with an 0.7 Nammonia/methanol solution to give the title compound (340.1 mg, 74.7%).

Example 9 (JDM0641A)

N-[1-[2-[2-(2-methoxyethoxyethoxy]ethyl]-3,5-dimethyl-pyrazol-4-yl]-2-[2-methoxy-4-(1,3,5-trimethylpyrazol-4-yl)anilino]-5,6-dihydropyrimido[45-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate B as starting material. The carboxylic acid wassubsequently reacted with Intermediate C in an analogous manner asdescribed for Example 8d. Purification was performed using preparativeHPLC to afford the title compound (19.5 mg, 42.6%). Data: LCMS (B)R_(t): 10.946 min; m/z 684.7 (M+H).

Intermediate D (JGS88/92)

2-Methoxy-4-(4-methylpiperazin-1-yl)aniline

This compound was prepared in an analogous manner as described forIntermediate A, starting from N-methylpiperazine and2-methoxy-4-fluoronitrobenzene to afford the title compound (1.38 g,94%).

Example 10 (JDM0443A)

N-[1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]-3,5-dimethyl-pyrazol-4-yl]-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid chloride, usingthe same sequence of reactions, as described for Intermediate 2, usingIntermediate D as starting material. The acid chloride was subsequentlyreacted with Intermediate C according to procedures described in Example8d. Purification was performed using preparative HPLC to afford thetitle compound (11.6 mg, 28.6%). Data: LCMS (B) R_(t): 6.985 min; m/z674.3 (M+H)⁺.

Example 11 (JGS79C)

N-(2,6-diethylphenyl)-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding ester, using the samesequence of reactions as described for Intermediate 2a usingIntermediate D as starting material. LiHDMS (1M in THF/ethylbenzene, 412μL, 0.412 mmol) was added to a cold (0° C.) solution of2,6-diethylaniline (50.8 μL, 0.31 mmol) in THF (1 mL). After 15 minutesof stirring at 0° C., ethyl2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(48 mg, 0.103 mmol) in THF (2 mL) was added drop-wise to the reactionmixture and stirring was continued for 90 min at 0° C. Additional LiHMDS(100 μL) was added drop-wise at room temperature and stirring wascontinued for 2 hours at room temperature. The reaction mixture wasquenched with 20 mL saturated solution of ammonium chloride andextracted with ethyl acetate. The combined organic layers were washedwith water, brine, dried over sodium sulfate, filtered and concentratedunder reduced pressure. Purification was performed using preparativeHPLC to afford the title compound (13.5 mg, 23.2%). Data: LCMS (C)R_(t): 12.686 min; m/z 566.4 (M+H)⁺.

Intermediate E (/WITJ437WITJ438/WITJ440)

2-(Difluoromethoxy)-4-(4-methylpiperazin-1-yl)aniline

To a solution of 5-fluoro-2-nitro-phenol (500 mg, 3.18 mmol) in DMF (6ml) was added sodium 2-chloro-2,2-difluoro-acetate (970 mg, 6.36 mmol)and disodium carbonate (405 mg, 3.82 mmol). The reaction mixture wasstirred at 100° C. for 3.5 hours and subsequently at room temperaturefor 3 days. A 4M HCl-solution was added until a clear solution wasobtained and the mixture was stirred for 2 h at room temperature. Thereaction mixture was diluted with water and extracted with EtOAc. Thecombined organic layers were washed with 1M NaOH-solution, brine, driedover sodium sulphate, filtered and concentrated in vacuo. The residuewas purified by column chromatography (heptane/ethyl acetate=10/0 to 8/2v/v %) to afford 2-(difluoromethoxy)-4-fluoro-1-nitro-benzene (493 mg,75%).

The title compound was prepared in an analogous manner as described forIntermediate A, starting from N-methylpiperazine and2-(difluoromethoxy)-4-fluoro-1-nitro-benzene to afford 180 mg (80%).

Intermediate F (JDM300/WITJ410/WITJ411/WITJ413)

3,5-Diethyl-1H-pyrazol-4-amine (a) 3,5-Diethyl-1H-pyrazole

To a solution of 3,5-heptanedione (2 g, 15.6 mmol) and hydrazine hydrate(0.77 g, 15.8 mmol) in water (10 mL) was added acetic acid (1 drop) andthe reaction mixture was heated to reflux for 1 h. The reaction mixturewas then cooled, and concentrated under reduced pressure to provide 1.8g of the title compound. This compound was used directly in the nextstep without purification.

(b) 3,5-Diethyl-4-nitro-1H-pyrazole

To a cold (0° C.) mixture of 3,5-diethyl-1H-pyrazole (1.8 g, 14.5 mmol)and concentrated sulphuric acid (1.5 ml) was added slowly, undervigorous stirring, fuming HNO₃ (4.35 ml). The reaction mixture wasstirred overnight at 60° C. The mixture was subsequently cooled to roomtemperature, then carefully added to an ice-cold saturated solution ofsodium bicarbonate and stirred for 15 min. The mixture was thenextracted three times with EtOAc and combined organic layers were washedwith brine, dried over sodium sulphate, filtered and evaporated in vacuoto give: 2.52 g 3,5-diethyl-4-nitro-1H-pyrazole.

(c) 3,5-Diethyl-1H-pyrazol-4-amine (Intermediate F)

The title compound was prepared in an analogous manner as described forIntermediate C, starting from 3,5-diethyl-4-nitro-1H-pyrazole to give3,5-diethyl-1H-pyrazol-4-amine (174 mg, 71%).

Example 12 (WITJ453B)

N-(3,5-diethyl-1H-pyrazol-4-yl)-2-[2-(difluoromethoxy)-4-(4-methylpiperazin-1-yl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding carboxylic acid, usingthe same sequence of reactions as described for Intermediate 2b, usingIntermediate E as starting material. The carboxylic acid wassubsequently reacted with Intermediate F in an analogous manner asdescribed for Example 8d. Purification was performed using preparativeHPLC to afford the title compound (9.6 mg, 23%). Data: LCMS (B) R_(t):8.864 min; m/z 592.3 (M+H)⁺.

Intermediate G (JDM617/JDM622/JDM630)

3,5-Diethyl-1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]pyrazol-4-amine

The title compound was prepared in an analogous manner as described forIntermediate C, starting from 3,5-diethyl-4-nitro-1H-pyrazole(Intermediate Fb) and triethylene glycol monomethyl ether to give 660 mgof 3,5-diethyl-1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]pyrazol-4-amine(41.7%).

Intermediate M (WITJ461/WITJ4587)

Benzyl 4-(4-amino-3-methoxy-phenyl)piperazine-1-carboxylate

This compound was prepared in an analogous manner as described forIntermediate A, starting from benzyl piperazine-1-carboxylate and2-methoxy-4-fluoronitrobenzene to afford the title compound (1.2 g,95%).

Example 13 (JDM0684A)

N-[3,5-diethyl-1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]pyrazol-4-yl]-2-[2-methoxy-4-[4-(2-methoxyacetyl)piperazin-1-yl]anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding amine (prepared asdescribed for Example 7 starting from Intermediate 1 and Intermediate M)and methoxyacetic acid, using standard HATU-coupling procedures asdescribed in Example 8d. Purification was performed using preparativeHPLC to afford the title compound (17.8 mg, 57.1%). Data: LCMS (B)R_(t): 9.908 min; m/z 760.8 (M+H)⁺.

Intermediate H (JDM221/JDM0222/JDM393)

1-(2-Methoxyethyl)-3,5-dimethyl-pyrazol-4-amine (a)1-(2-Methoxyethyl)-3,5-dimethyl-4-nitro-pyrazole

To a solution of 3,5-dimethyl-4-nitro-1H-pyrazole (2.5 g, 17.7 mmol) andcaesium carbonate (6.06 g, 18.6 mmol) in DMF (50 mL) was added2-bromoethyl methyl ether (2.59 g, 1.75 mL, 18.6 mmol). The mixture washeated at 100° C. for 3.5 h. After cooling to room temperature, themixture was poured into water and extracted with ethyl acetate (3×50mL). The combined organic layers were washed with brine (50 mL), driedover sodium sulfate, filtered and concentrated under reduced pressure.The residue was purified by column chromatography (EtOAc/heptanes=1/4v/v %) to afford 1-(2-methoxyethyl)-3,5-dimethyl-4-nitro-pyrazole (2.66g, 75.4%) as a white crystalline solid.

(b) 1-(2-Methoxyethyl)-3,5-dimethyl-pyrazol-4-amine

1-(2-Methoxyethyl)-3,5-dimethyl-4-nitro-pyrazole (245 mg, 1.22 mmol) wasdissolved in methanol (25 mL). The resulting solution was hydrogenatedusing a H-Cube continuous-flow hydrogenation reactor, 10% Pd/C, at 30°C., 8-10 bar, 1 mL/min, full H₂ modus. The resulting solution wasconcentrated in vacuo to yield 208 mg (quant. yield) of the titlecompound as a light-brown oil.

Intermediate I (JDM464/JDM450)

3,5-Diethyl-1-[2-(2-methoxyethoxy)ethyl]pyrazol-4-amine

The title compound was prepared in an analogous manner as described forIntermediate H, starting from 3,5-diethyl-4-nitro-1H-pyrazole(Intermediate Fb) and 1-bromo-2-(2-methoxyethoxy)-ethane to give 290 mgof 3,5-diethyl-1-[2-(2-methoxyethoxy)ethyl]pyrazol-4-amine (72.2%.).

Example 14 (JDM0466A)

N-[3,5-diethyl-1-[2-(2-methoxy)ethyl]pyrazol-4-yl]-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid chloride, usingthe same sequence of reactions, as described for Intermediate 2, usingIntermediate D as starting material. The acid chloride was subsequentlyreacted with Intermediate I according to procedures described in Example7. Purification was performed using preparative HPLC to afford the titlecompound (22 mg, 54.4%). Data: LCMS (B) R_(t): 7.845 min; m/z 658.3(M+H).

Intermediate J (WITJ277/WITJ84)

tert-Butyl 4-(4-amino-3-methoxy-phenyl)piperazine-1-carboxylate

This compound was prepared in an analogous manner as described inIntermediate A, starting from tert-butyl piperazine-1-carboxylate and2-methoxy-4-fluoronitrobenzene to afford the title compound (245 mg,91%).

Example 15 (WITJ349B)

N-[1-(2-m ethoxyyl-3,5-dimethyl-pyrazol-4-yl]-2-(2-methoxy-4-piperazin-1-yl-anilino)-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

Ethyl 2-chloro-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(Intermediate 1, 296 mg, 1.07 mmol), tert-Butyl4-(4-amino-3-methoxy-phenyl)piperazine-1-carboxylate (Intermediate J,328 mg, 1.07 mmol) and cesium carbonate (1.39 g, 4.27 mmol) weresuspended in dioxane (25 mL). Nitrogen was bubbled through the mixtureat 30° C. for 5 minutes followed by the addition of9,9-bis-dimethyl-4,5-bis(diphenylphosphino)xanthene (62 mg, 0.11 mmol)and tris(dibenzylideneacetone)dipalladium(0) (49 mg, 53 μmol). Thereaction mixture was stirred at 80° C. for 20 hours under a flow ofnitrogen gas.

Ethyl acetate/water/brine (1/1/1 v/v %, 50 mL) were added to thereaction mixture and stirring was continued for 15 min. After filtrationover Decalite® the water layer was separated and extracted with ethylacetate (2×20 mL). The combined organic layers were subsequently washedwith water (40 mL), brine (20 mL), dried over sodium sulphate, filteredand concentrated in vacuo. The crude product was purified by columnchromatography on silica (Heptane/Ethyl acetate=1/0 to 0/1 v/v %) toethyl2-[4-(4-tert-butoxycarbonylpiperazin-1-yl)-2-methoxy-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate(115 mg, 20%).

The thus obtained ethyl ester was subsequently hydrolysed usingconditions described for Intermediate 2b. The sodium salt of thecorresponding carboxylic acid was subsequently reacted with IntermediateH in an analogous manner as described for Example 8d. Afterde-protection of the Boc-group, purification was performed usingpreparative HPLC to afford the title compound (5.2 mg, 28%). Data: LCMS(B) R_(t): 8.140 min; m/z 572.3 (M+H)⁺.

Intermediate K (JDM25/JDM302)

2-Methoxy-4-[1-methyl-4-piperidyloxy]aniline (a)4-(3-Methoxy-4-nitro-phenoxy)-1-methyl-piperidine

To a solution of 4-fluoro-2-methoxy-1-nitro-benzene (750 mg, 4.38 mmol)in toluene (10 mL) were added 10 mL of a 25% KOH-solution,4-hydroxy-N-methylpiperidine (1009 mg, 8.76 mmol) and tetra-n-butylammonium bromide (282 mg, 0.876 mmol). The mixture was heated at 60° C.o/n. The reaction mixture was then diluted with ethyl acetate and thewater layer was extracted with ethyl acetate. The combined organiclayers were washed with brine, dried over sodium sulfate and evaporated.The residue was purified by flash chromatography on silica gel(dichloromethane/methanol=99/1 to 9/1 v/v %) to obtain the titlecompound. (650 mg, 55.7%)

(b) 2-Methoxy-4-[(1-methyl-4-piperidyl)oxy]aniline (Intermediate K)

10% Pd/C (20 mg) was added as a suspension in ethanol to a solution of4-(3-methoxy-4-nitro-phenoxy)-1-methyl-piperidine (200 mg, 0.75 mmol) inethanol (5 mL). The resulting mixture was stirred for 15 min at roomtemperature. Ammonium formate (473 mg, 7.5 mmol) was added and thereaction mixture was stirred for 1 hour at reflux under nitrogenatmosphere. The reaction mixture was cooled to room temperature andfiltered over Decalite®. The filtrate was concentrated in vacuo, afterwhich dichloromethane was added and the organic phase was washed with 5%solution of NaHCO₃. The organic phase was dried over sodium sulfate,filtered and concentrated in vacuo to yield2-methoxy-4-[(1-methyl-4-piperidyl)oxy]aniline (169.5 mg, 95.6%).

Intermediate L (JDM221J/JDM0222)

3,5-Dimethyl-1H-pyrazol-4-amine

The title compound was prepared in an analogous manner as described forIntermediate Hb, starting from 3,5-dimethyl-4-nitro-1H-pyrazole to give110 mg 3,5-dimethyl-1H-pyrazol-4-amine (quant.).

Example 16 (JDM323A)

N-(3,5-dimethyl-1H-pyrazol-4-≡(1-2-[2-methoxy-4-[(1-methyl-4-piperidyl)oxy]anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid chloride, usingthe same sequence of reactions, as described for Intermediate 2, usingIntermediate K as starting material. The acid chloride was subsequentlyreacted with Intermediate L according to procedures described in Example7. Purification was performed using preparative HPLC to afford the titlecompound (14.1 mg, 37%). Data: LCMS (B) R_(t): 7.902 min; m/z 543.2(M+H).

Example 17 (WITJ0529B)

N-(2,6-dimethylphenyl)-2-[2-methoxy-4-[4-(2-methoxyacetyl)piperazin-1-yl]anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding amine (prepared asdescribed for Example 7 starting from Intermediate 1 and Intermediate M)and methoxyacetic acid, using standard HATU-coupling procedures asdescribed in Example 8d. Purification was performed using preparativeHPLC to afford the title compound (10 mg, 49%). Data: LCMS (B) R_(t):12.973 min; m/z 596.3 (M+H)⁺.

Intermediate 3

Ethyl 2-chloropyrimido[4,5-e]indolizine-7-carboxylate (a) Ethyl2-methoxypyrimido[4,5-e]indolizine-7-carboxylate (JGS362)

DDQ (1.53 g, 6.76 mmol) was added to a stirred solution of ethyl2-methoxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate (1.54 g,5.63 mmol) in DCM (50 mL). The reaction mixture stirred for 3 days atroom temperature. An additional amount of 200 mg DDQ was added and thereaction mixture was stirred for another 7 days at room temperature. Themixture was filtered and concentrated in vacuo to a small volume. Thecrude product was purified by column chromatography on silica(heptane/ethyl acetate=I/O to 1/1 v/v %) to yield the title compound(750 mg, 50%).

(b) Ethyl 2-hydroxypyrimido[4,5-e]indolizine-7-carboxylate (JGS377)

Sodium iodide (1.24 g, 8.29 mmol) was added to a stirred solution ofethyl 2-methoxy-pyrimido[4,5-e]indolizine-7-carboxylate (750 mg, 2.76mmol) in acetonitrile (19 mL). A solution of trimethylsilyl chloride(896 mg, 1.05 mL) in acetonitrile (3 mL) was added drop-wise to thereaction mixture. The mixture was stirred at room temperature o/n.Additional sodium iodide (3.33 g) TMS-Cl (2.4 g, 2.8 mL) in acetonitrile(6 mL) were added drop-wise and the reaction was stirred for 3 days atroom temperature. The mixture was concentrated under reduced pressure.The residue was suspended in 200 mL DCM/MeOH (4/1) and extracted with amixture of a saturated solution of sodium thiosulfate (50 mL) and water(100 mL). The water layer was extracted with DCM/MeOH (4/1, 2×150 mL).The combined organic layers were dried over sodium sulfate, filtered andthe solvent was removed under reduced pressure to give a solid. Thesolid was triturated in boiling ethyl acetate (50 mL). After cooling thesolid was stirred 1h at room temperature and filtered. The residue wasdried at 40° C. under vacuum to give 1.0 g crude ethyl2-hydroxy-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxylate (quant.yield).

(c) Ethyl 2-chloropyrimido[4,5-e]indolizine-7-carboxylate (Intermediate3) (JGS380)

N,N-Dimethylaniline (47 mg, 50 μL, 1.50 mmol) was added to a solution ofethyl 2-hydroxypyrimido[4,5-e]indolizine-7-carboxylate (1.0 g, 3.89mmol) in acetonitrile (30 mL). A solution of phosphorous(V) oxychloride(2.99 g, 1.81 mL, 19.5 mmol) in acetonitrile (4 mL) was added drop-wiseto the reaction mixture. The brown/red suspension was heated to 65° C.for 4 hours. After cooling, the mixture was slowly poured in a stirredmixture of 25% aq. ammonia (50 mL) and ice-water (100 mL) keeping thetemperature below 10° C. After stirring for another 15 minutes themixture was extracted with ethyl acetate. The combined organic layerswere subsequently washed with water (50 mL), 0.2 N HCl (50 mL), brine(25 mL), dried (Na₂SO₄), filtered and concentrated in vacuo. The crudeproduct was purified by column chromatography on silica (heptane/ethylacetate=1/0 to 1/1 v/v %) to yield 200 mg of the title compound.

Example 18 (WITJ490B)

N-(2,6-dimethylphenyl)-2-[2-methoxy-4-[(1-methyl-4-piperidyloxy]anilino]pyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid chloride, usingthe same sequence of reactions as described for Intermediate 2, startingfrom Intermediate 3 and Intermediate K as starting material. The acidchloride was subsequently reacted with 2,6-dimethylaniline according toprocedures described in Example 7. Purification was performed usingpreparative HPLC to afford the title compound (30 mg, 45%). Data: LCMS(B) R_(t): 12.491 min; m/z 551.3 (M+H)⁺.

Intermediate N (JDM618/JDM626/JDM634)

1-[2-(2-Ethoxyethoxy)ethyl]-3,5-diethyl-pyrazol-4-amine (a)2-(2-Ethoxyethoxylethyl 4-methylbenzenesulfonate

To a solution of di(ethylene glycol)ethyl ether (4.92 ml, 36.2 mmol) in15 mL of THF, cooled at 0° C., was added NaOH (2.46 g. 61.5 mmol)dissolved in 15 mL of water with vigorous stirring. To this mixture wasadded dropwise a solution of tosyl chloride (8.28 g. 43.4 mmol) in 15 mLof THF over 10 min at 0° C. The reaction mixture was then raised to rtand stirred for 1 h under nitrogen. The mixture was then extracted twicewith 50 mL of diethyl ether, and the organic layer was washed with 1 Maq NaOH and water and dried over sodium sulfate. Solvent was removedunder reduced pressure to yield 2-(2-ethoxyethoxy)ethyl4-methylbenzenesulfonate as a colorless liquid (10 g. 95.8%).

(b) 1-[2-(2-Ethoxyethoxyl)ethyl]-3,5-dimethyl-4-nitro-pyrazole

To a solution of 3,5-dimethyl-4-nitro-1H-pyrazol (1 g. 7.08 mmol) andcesium carbonate (2.31 g. 7.08 mmol) in DMF (10 mL) was added2-(2-ethoxyethoxy)ethyl 4-methylbenzenesulfonate (2.04 g. 7.08 mmol).The mixture was heated at 100° C. for 1 h. After cooling to roomtemperature, the mixture was poured into water/brine and extracted withethyl acetate (100 mL). The combined organic layer was washed withbrine, dried over sodium sulfate, filtered and concentrated underreduced pressure to yield 1.69 g of the title compound (92.8%).

(c) 1-[2-(2-Ethoxyethoxy)ethyl]-3,5-dimethyl-pyrazol-4-amine(Intermediate N)

To a stirred solution of1-[2-(2-ethoxyethoxy)ethyl]-3,5-dimethyl-4-nitro-pyrazole (1.69 g, 6.57mmol) in methanol (25 mL) was added a suspension of 10% Pd on charcoal(200 mg) in ethanol (1 mL). The reaction mixture was stirred at roomtemperature for 15 min under a nitrogen atmosphere. Then, ammoniumformate (4.14 g, 65.7 mmol) was added and the reaction mixture washeated to reflux temperature for 15 min. The reaction mixture wascooled, filtered over Decalite® and concentrated in vacuo. The residuewas dissolved in methanol and then filtered over an SCX-2 column. Afterrinsing the column with methanol, the desired product was eluted with an0.7N ammonia/methanol solution. The resulting eluate was concentrated invacuo to give the title compound (520 mg, 34.8%).

Example 19 (JDM640A)

N-[1-[2-(2-ethoxyethoxy)ethyl]-3,5-dimethyl-pyrazol-4-yl]-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate D as starting material. The carboxylic acid wassubsequently reacted with Intermediate N in an analogous manner asdescribed for Example 8d. Purification was performed using preparativeHPLC to afford the title compound (32.3 mg, 53.9%). Data: LCMS (B)R_(t): 7.432 min; m/z 644.6 (M+H)⁺.

Example 20 (JDM677A)

N-[1-[2-[2-(2-methoxyethoxy)ethoxyethyl]-3,5-dimethyl]-pyrazol-4-yl]-2-[2-methoxy-4-[4-(2-methoxyacetyl)piperazin-1-yl]anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate M as starting material. The carboxylic acid wassubsequently reacted with Intermediate C in an analogous manner asdescribed for Example 8d. The corresponding amine was obtained afterdeprotection of the Cbz-group and methoxyacetic acid was introduced,using standard HATU-coupling procedures as described in Example 8d.Purification was performed using preparative HPLC to afford the titlecompound (19.0 mg, 63.4%). Data: LCMS (B) R_(t): 8.815 min; m/z 732.7(M+H)⁺.

Example 21

N-[3,5-diethyl-1-[2-(2-methoxyethoxyethyl]pyrazol-4-yl]-2-[2-methoxy-4-[4-(3-methylazetidine-3-carbonyl)piperazin-1-yl]anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate M as starting material. The carboxylic acid wassubsequently reacted with Intermediate I in an analogous manner asdescribed for Example 8d. The corresponding amine was obtained afterdeprotection of the Cbz-group and1-(tert-butoxycarbonyl)-3-methylazetidine-3-carboxylic acid wasintroduced, using standard HATU-coupling procedures as described inExample 8d. Purification was performed, after deprotection of theBoc-group, using preparative HPLC to afford the title compound (16.3 mg,55%). Data: LCMS (B) R_(t): 8.107 min; m/z 741.8 (M+H)⁺.

Intermediate O (JDM618/JDM625/JDM633)

1-[2-(2-Ethoxyethoxy)ethyl]-3,5-diethyl-pyrazol-4-amine

The title compound was prepared in an analogous manner as described forIntermediate N, starting from 3,5-diethyl-4-nitro-1H-pyrazole(Intermediate Fb) and di(ethylene glycol)ethyl ether to give 550 mg of1-[2-(2-ethoxyethoxy)ethyl]-3,5-diethyl-pyrazol-4-amine (79.8%.).

Example 22 (JDM713A)

N-[3,5-diethyl-1-[2-(2-methoxyethoxy)ethyl]pyrazol-4-yl]-2-[2-methoxy-4-[4-(3-methylazetidine-3-carbonyl)piperazin-1-yl]anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate M as starting material. The carboxylic acid wassubsequently reacted with Intermediate O in an analogous manner asdescribed for Example 8d. The corresponding amine was obtained afterdeprotection of the Cbz-group and Boc-N-ethyl-glycine was introduced,using standard HATU-coupling procedures as described in Example 8d.Purification was performed, after deprotection of the Boc-group, usingpreparative HPLC to afford the title compound (15 mg, 53.1%). Data: LCMS(B) R_(t): 8.619 min; m/z 743.8 (M+H)⁺.

Example 23 (JDM697A)

N-[1-[2-(2-ethoxyethoxy)ethyl]-3,5-diethyl-pyrazol-4-yl]-2-[2-methoxy-4-[4-(2-methoxyacetyl)piperazin-1-yl]anilino]-5,6-dihydropyrimido[45-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate M as starting material. The carboxylic acid wassubsequently reacted with Intermediate O in an analogous manner asdescribed for Example 8d. The corresponding amine was obtained afterdeprotection of the Cbz-group and methoxyacetic acid was introduced,using standard HATU-coupling procedures as described in Example 8d.Purification was performed using preparative HPLC to afford the titlecompound (17.0 mg, 61.4%). Data: LCMS (B) R_(t):10.554 min; m/z 730.7(M+H)⁺.

Example 24 (JDM636A)

N-[3,5-diethyl-1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]pyrazol-4-yl]-2-[2-methoxy-4-(4-methylpiperazin-1-yl)anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate D as starting material. The carboxylic acid wassubsequently reacted with Intermediate G in an analogous manner asdescribed for Example 8d. Purification was performed using preparativeHPLC to afford the title compound (22.5 mg, 34.5%). Data: LCMS (B)R_(t): 7.879 min; m/z 702.7 (M+H).

Example 25 (JDM703A)

N-[3,5-diethyl-1-[2-[2-(2-methoxyethoxy)ethoxy]ethyl]pyrazol-4-yl]-2-[4-[4-[2-(ethylamino)acetyl]piperazin-1-yl]-2-methoxy-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate M as starting material. The carboxylic acid wassubsequently reacted with Intermediate G in an analogous manner asdescribed for Example 8d. The corresponding amine was obtained afterdeprotection of the Cbz-group and Boc-N-ethyl-glycine was introduced,using standard HATU-coupling procedures as described in Example 8d.Purification was performed, after deprotection of the Boc-group, usingpreparative HPLC to afford the title compound (18.4 mg, 59.4%). Data:LCMS (B) R_(t): 8.194 min; m/z 773.8 (M+H)⁺.

Example 26 (JDM709A)

2-[4-[4-[(2R)-azetidine-2-carbonyl]piperazin-1-yl]-2-methoxy-anilino]-N-[3,5-diethyl-1-[2-(2-methoxyethoxy)ethyl]pyrazol-4-yl]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid, using the samesequence of reactions, as described for Intermediate 2b, usingIntermediate M as starting material. The carboxylic acid wassubsequently reacted with Intermediate I in an analogous manner asdescribed for Example 8d. The corresponding amine was obtained afterdeprotection of the Cbz-group and (R)—N-Boc-azetidine-2-carboxylic acidwas introduced, using standard HATU-coupling procedures as described inExample 8d. Purification was performed, after deprotection of theBoc-group, using preparative HPLC to afford the title compound (16.8 mg,57.7%). Data: LCMS (B) R_(t): 8.017 min; m/z 727.9 (M+H)⁺.

Example 27 (JDM666A)

N-(2,6-dimethylphenyl-2-[4-[4-[2-ethylamino)acetyl]piperazin-1-yl]-2-methoxy-anilino]-5,6-dihydropyrimido[4,5-e]indolizine-7-carboxamide

This compound was prepared from its corresponding acid chloride, usingthe same sequence of reactions, as described for Intermediate 2, usingIntermediate M as starting material. The acid chloride was subsequentlyreacted with 2,6-dimethylaniline in an analogous manner as described forExample 7. The corresponding amine was obtained after deprotection ofthe Cbz-group and Boc-N-ethyl-glycine was introduced, using standardHATU-coupling procedures as described in Example 8d. Purification wasperformed, after deprotection of the Boc-group, using preparative HPLCto afford the title compound (1 mg, 13.2%). Data: LCMS (B) R_(t): 9.388min; m/z 609.6 (M+H)⁺.

Example 28 (JGS0715B)

N-cyclopropyl-4-[6-(2,3-difluoro-4-methoxy-phenoxy)-8-(3,3,3-trifluoropropylamino)imidazo[1,2-b]pyridazin-3-yl]-2-methyl-benzamide

This compound was prepared as described in WO 2014/131739 A1.Purification was performed using preparative HPLC to afford the titlecompound (30 mg). Data: LCMS (B) R_(t): 14.958 min; m/z 562.5 (M+H)⁺.

Example 29 (JDM943L)

(2R)-2-(4-fluorphenyl)-N-[4-[2-(2-methoxy-4-methylsulfonyl-anilino)-[1,2,4]triazolo-[1,5-a]pyridin-6-yl]phenyl]propanamide

This compound was prepared as described in WO 2014/009219 A1.Purification was performed using preparative HPLC to afford the titlecompound (107.1 mg). Data: LCMS (B) R_(t): 13.703 min; m/z 558.0 (M−H)⁻.

Example 30 (JDM969A)

1-[4-[[4-(2-Isopropylsulfonylanilino)-1H-pyrrolo[2,3-b]pyridin-6-yl]amino]-3-methoxy-phenyl]piperidin-4-ol(Mps1-IN-1)

This compound was purchased from Tocris.

Example 31 (JDM969B)

4-[[4-Amino-6-(tert-butylamino)-5-cyano-2-pyridyl]amino]benzamide (TCMps1 12)

This compound was purchased from Tocris.

TTK Enzyme Assay

The inhibitory activity of compounds on biochemically purifiedfull-length TTK (Life Technologies, Madison, Wis., U.S.A.) wasdetermined in the IMAP® assay (Molecular Devices, Sunnyvale, Calif.,U.S.A.). Compounds were dissolved in 100% dimethylsulfoxide (DMSO). Atthe day of the experiment, the compound stock was diluted in 3.16 foldsteps in 100% DMSO, to obtain a 10-point dilution series, followed byfurther dilution in IMAP reaction buffer, which consists of 10 mMTris-HCl, pH 7.5, 10 mM MgCl₂, 0.01% Tween-20, 0.1% NaN₃ and 1 mMfreshly prepared dithiothreitol. Compound solution was mixed with anequal volume of full-length TTK enzyme in IMAP reaction buffer. Afterpre-incubation of 1 hour in the dark at room temperature,fluorescein-labeled MBP-derived substrate peptide (Molecular Devices)was added and ATP to start the reaction. Final enzyme concentration was3.9 nM, final substrate concentration 50 nM, and final ATP concentrationwas 5 μM. The reaction was allowed to proceed for 2 hours at roomtemperature in the dark. The reaction was stopped by quenching with IMAPprogressive binding solution according to the protocol of themanufacturer (Molecular Devices). Fluorescein polarization was measuredon an Envision multimode reader (Perkin Elmer, Waltham, Mass., USA).Dose-response curves were fitted to a four-parameter logarithmicequation in XLfit™ 5 (ID Business Solutions, Ltd., Guildford, U.K.).Table 3 show the half-maximum inhibitory potency of a number of TTKinhibitors from different chemical classes in an enzyme assay for TTK.

TABLE 3 Activity of small molecule TTK inhibitors in TTK enzyme assay.Example nr. IC₅₀ (nM) Example 1 2.6 Example 2 4.1 Example 3 0.63 Example4 0.49 Example 5 0.65 Example 6 0.6 Example 7 0.7 Example 8 0.6 Example9 0.73 Example 10 0.93 Example 11 0.6 Example 12 0.97 Example 13 0.43Example 14 0.98 Example 15 1.1 Example 16 1.0 Example 17 1.0 Example 181.4 Example 19 1.14 Example 20 0.62 Example 21 0.96 Example 22 0.71Example 23 0.41 Example 24 0.91 Example 25 0.43 Example 26 0.66 Example27 0.41 Example 28 0.79 Example 29 2.55 Example 30 37.5 Example 31 7.0

In order to identify genomic biomarkers that correlate with thesensitivity of cancer cells to the TTK inhibitors, the compounds weretested in proliferation assays with sixty-six different, geneticallywell-characterized cancer cell lines.

Statistical analysis of the anti-proliferative activity of theinhibitors with the presence of specific cancer gene mutations in thecell lines revealed that TTK inhibitors preferentially kill cells thatharbor mutation in the CTNNB1 gene known to be involved in regulation ofthe stability of the CTNNB1-encoded protein β-catenin.

FIG. 2 shows the volcano plot of the Anova analysis of Examples 5, 8, 9,12, 13 and 17. To verify if a TTK inhibitor was significantly morepotent in cell lines expressing mutant CTNNB1 in comparison to celllines not harboring mutation in CTNNB1, a one-sided Student's t-test wascarried out. Table 4 shows the difference in sensitivity (ΔpIC₅₀) of anumber of representative TTK inhibitors from different chemical classes.A negative ΔpIC₅₀ value indicates that CTNNB1 mutant cell lines are moresensitive to the inhibitor than cell lines not harboring mutations inthe regulatory domain of the CTNNB1 gene (Table 2). A p value <0.05indicates that the difference is significant.

TABLE 4 Difference in sensitivity of CTNNB1-mutant and non-mutant celllines for TTK inhibitors pIC50 average¹ significance Example nr. Wildtype³ Mutant³ Δ pIC50 p-value² Example 1 6.15 6.78 −0.63 5.0E−03 Example2 5.73 5.98 −0.25 3.6E−02 Example 3 5.68 6.01 −0.33 2.4E−02 Example 47.21 7.64 −0.43 3.3E−02 Example 5 7.06 7.60 −0.54 1.2E−02 Example 6 6.937.64 −0.71 8.8E−03 Example 7 7.47 7.92 −0.45 2.6E−02 Example 8 7.38 7.96−0.57 1.3E−02 Example 9 7.50 8.18 −0.68 1.9E−02 Example 10 6.86 7.41−0.55 1.4E−02 Example 11 6.99 7.48 −0.49 2.7E−02 Example 12 6.96 7.65−0.69 6.5E−03 Example 13 7.82 8.60 −0.79 7.1E−03 Example 14 7.47 8.01−0.54 1.8E−02 Example 15 6.60 7.28 −0.69 3.9E−03 Example 16 6.46 7.22−0.76 4.6E−03 Example 17 7.72 8.33 −0.61 1.7E−02 Example 18 7.53 8.13−0.60 2.9E−02 Example 19 7.20 7.72 −0.53 9.3E−03 Example 20 7.28 7.90−0.2 8.7E−03 Example 21 7.19 7.98 −0.79 1.2E−02 Example 22 7.70 8.34−0.65 9.1E−03 Example 23 7.97 8.55 −0.59 2.0E−02 Example 24 7.59 8.09−0.50 2.5E−02 Example 25 7.62 8.37 −0.75 3.2E−03 Example 26 7.28 7.96−0.68 9.9E−03 Example 27 7.60 8.17 −0.57 1.9E−02 Example 28 7.00 7.47−0.47 1.1E−02 Example 29 6.40 6.72 −0.31 1.8E−02 Example 30 5.17 5.39−0.23 1.0E−01 Example 31 6.02 6.36 −0.34 2.8E−02 ¹defined as-¹⁰log IC₅₀(in M) ²one-side student T-test, heteroscedastic ³referring to CTNNB1gene

In order to verify that the presence of a mutated CTNNB1 gene copy issufficient to confer increased sensitivity to TTK inhibitors,proliferation assays were carried out with parental HCT116 cells(S45del/+) and an isogenic derivative lacking mutated CTNNB1 (−/+).Table 5 summarizes the difference in sensitivity of a number ofrepresentative TTK inhibitors from different chemical classes in theisogenic cell line in comparison to parental HCT116 cells. A negativeΔpIC₅₀ or a negative Δefficacy indicates that HCT116 parental cells,expressing mutant CTNNB1 (S45del/+) are more sensitive to the inhibitorthan the isogenic derivative, in which the mutated CTNNB1 gene has beenremoved (−/+). Thus, inhibitors with a negative ΔpIC50 or a negativeΔefficacy better inhibit the cell line where mutant CTNNB1 signaling ispresent.

TABLE 5 Difference in sensitivity for TTK inhibitors in HCT116 cellsexpressing either a mutated copy of the CTNNB1 gene or not. ΔpIC₅₀Δefficacy Example 5 −0.17 −26 Example 8 −0.29 −11 Example 12 −0.14 −18Example 13 −0.01 −27 Example 17 −0.12 −16 Example 20 −0.20 −34

1. A method for identifying a tumor in a human individual or an animalthat is susceptible to treatment with a TTK inhibitor, the methodcomprising: a) providing a sample of a tumor; b) determining thepresence of a mutated CTNNB1 gene in the tumor sample, wherein themutation is located in exon 3 of CTNNB1, whereby the presence of amutated CTNNB1 gene indicates that the tumor is susceptible to treatmentwith a TTK inhibitor.
 2. The method according to claim 1, wherein themutation is a missense mutation or a deletion of the serine residuecorresponding to codon 33 of CTNNB1.
 3. The method according to claim 1,wherein the mutation is a missense mutation or a deletion of thethreonine residue corresponding to codon 41 of CTNNB1.
 4. The methodaccording to claim 1, wherein the mutation is a missense mutation or adeletion of the serine residue corresponding to codon 45 of CTNNB1.
 5. Amethod for identifying a tumor in a human individual or an animal thatis susceptible to treatment with a TTK inhibitor, the method comprising:a) providing a sample of a tumor; b) determining the presence of amutated CTNNB1 protein in the tumor sample, wherein the mutation islocated in exon 3 of CTNNB1 whereby the presence of a mutated CTNNB1protein indicates that the tumor is susceptible to treatment with a TTKinhibitor.
 6. A method for identifying a tumor in a human individual oran animal that is susceptible to treatment with a TTK inhibitor, themethod comprising: a) providing a sample of a tumor; b) determining analtered expression of a CTNNB1 regulated gene, whereby an alteredexpression of a CTNNB1 regulated gene indicates that the tumor issusceptible to treatment with a TTK inhibitor.
 7. The method accordingto claim 1, wherein the TTK inhibitor is a chemical compound belongingto the class of compounds according to Formula I:

wherein: R¹ is selected from the group consisting of:

R¹¹ is H, halogen, (1-2C)alkyl, (2-3C)alkenyl, (2-3C)alkynyl,(1-2C)alkoxy or OC₂H₃, all alkyl and alkoxy groups optionally beingsubstituted with one or more halogen; R¹² is H, halogen, (1-2C)alkyl or(1-2C)alkoxy; R¹³ is R¹³¹CH₂, R¹³²⁰, R¹³³R¹³⁴N, R¹³⁵C(O), R¹³⁶S,R¹³⁶S(O), R¹³⁶S(O)(NH), R¹³⁷SO₂, (2-7C)heterocycloalkyl, or(1-5C)heteroaryl each heterocycloalkyl or heteroaryl optionally beingsubstituted with (1-2C)alkyl, fluoro, hydroxyl, oxo, (1-2C)alkoxy,(1-6C)alkylcarbonyl, (1-6C)alkylsulfonyl, (1-5C)alkoxycarbonyl,(1-6C)alkylaminocarbonyl, (3-6C)cycloalkylcarbonyl,(2-7C)heterocycloalkylcarbonyl or di[(1-2C)alkyl]amino, eachalkylcarbonyl, alkylsulfonyl, alkoxycarbonyl, alkylaminocarbonyl,cycloalkylcarbonyl or heterocycloalkylcarbonyl optionally beingsubstituted with (1-2C)alkyl, fluoro, hydroxyl, cyano, oxo or(1-2C)alkoxy; R¹³¹ is (1-6C)alkylcarbonylamino,(3-6C)cycloalkylcarbonylamino or (2-7C)heterocycloalkylcarbonylaminoeach optionally substituted with one or more groups selected from(1-2C)alkyl, fluoro, hydroxyl or (1-2C)alkoxy; R¹³² is (1-6C)alkyl,(3-6C)cycloalkyl, (2-7C)heterocycloalkyl, (6-10C)aryl or(1-5C)heteroaryl each optionally substituted with one or more groupsselected from (1-2C)alkyl, halogen, hydroxyl, (1-2C)alkoxy,di[(1-2C)alkyl]amino or (2-7C)heterocycloalkyl; R¹³³ is (1-6C)alkyl,(3-6C)cycloalkyl, (2-7C)heterocycloalkyl (1-6C)alkylcarbonyl,(1-5C)alkoxycarbonyl, (3-6C)cycloalkylcarbonyl or(2-7C)heterocycloalkylcarbonyl, each optionally substituted with one ormore groups selected from (1-2C)alkyl, halogen, hydroxyl or(1-2C)alkoxy, di[(1-2C)alkyl]amino or (2-7C)heterocycloalkyl; R¹³⁴ ishydrogen or (1-2C)alkyl; R¹³⁵ is (2-7C)heterocycloalkyl,(1-6C)alkylamino, di[(1-6C)alkyl]amino, (2-7C)heterocycloalkylamino or(3-6C)cycloalkylamino each optionally substituted with one or moregroups selected from (1-2C)alkyl, fluoro, hydroxyl, (1-2C)alkoxy,di[(1-2C)alkyl]amino, (2-7C)heterocycloalkyl, oxo, cyano or amino; R¹³⁶is (1-6C)alkyl, (3-6C)cycloalkyl, (2-7C)heterocycloalkyl each optionallysubstituted with one or more groups selected from (1-2C)alkyl, fluoro,hydroxyl or (1-2C)alkoxy; R¹³⁷ is (1-6C)alkyl, (3-6C)cycloalkyl,(2-7C)heterocycloalkyl, (1-6C)alkylamino, di[(1-6C)alkyl]amino,(2-7C)heterocycloalkylamino or (3-6C)cycloalkylamino, each optionallysubstituted with one or more groups selected from (1-2C)alkyl, fluoro,hydroxyl or (1-2C)alkoxy; R¹⁴ is H, halogen, (1-2C)alkyl or(1-2C)alkoxy; and R¹⁵ is H, halogen; in the above Formula I, R² isselected from the group consisting of:

R²¹ is H, halogen, (1-3C)alkyl, (1-2C)alkoxy, hydroxy(1-2C)alkyl,(3-4C)cycloalkyl, (2-3C)alkenyl or cyano; R²² is H, halogen, (1-2C)alkylor (1-2C)alkoxy; R²³ is H, halogen, (1-2C)alkyl, (1-2C)alkoxy, cyano orhydroxy; R²⁴ is H, halogen, (1-2C)alkyl or (1-2C)alkoxy; R²⁵ is H,halogen, (1-3C)alkyl, (1-2C)alkoxy, hydroxy(1-2C)alkyl,(3-4C)cycloalkyl, (2-3C)alkenyl or cyano; R²⁶ is H, (1-6C)alkyl,(3-6C)cycloalkyl, (2-5C)heterocycloalkyl,(1-2C)alkoxy[(2-4C)alkoxy]_(n)(1-6C)alkyl, wherein n represents aninteger of 1, 2, 3 or 4, all alkyl, heterocycloalkyl and(1-2C)alkoxy[(2-4C)alkoxy]_(n)(1-6C)alkyl groups optionally substitutedwith one or more groups selected from (1-2C)alkyl, (1-2C)alkoxy,hydroxyl, oxo, amino, (3-6C)cycloalkyl, di[(1-2C)alkyl]amino or(2-5C)heterocycloalkyl; and wherein in the above Formula I only one ofR²¹ and R²⁵ in R² can be H.
 8. The method according to claim 1, whereinthe TTK inhibitor is a chemical compound belonging to the class ofcompounds according to Formula II.

wherein: R¹ and R³ are independently selected from the group consistingof (6-10C)aryl and (1-5C)heteroaryl, wherein both groups optionally canbe substituted; R² is selected from the group consisting of (1-6C)alkyland (2-6C)alkenyl, wherein both groups optionally can be substituted; R⁴is selected from the group consisting of hydrogen and (1-6C)alkyl,wherein the alkyl group optionally can be substituted; and, R⁵ and R⁶are independently hydrogen or methyl.
 9. The method according to claim1, wherein the TTK inhibitor is a chemical compound belonging to theclass of compounds according to Formula III:

wherein, R¹ is selected from the group consisting of (1-6C)alkyl,halo(1-6C)alkyl, HO-(1-6C)alkyl, H₂N-(1-6C)alkyl, cyano(1-6C)alkyl,(1-6C)alkoxy(1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, (3-6C)cycloalkyl,(3-7C)heterocycloalkyl, (6-10C)aryl and (1-5C)heteroaryl, wherein thegroups optionally can be substituted; R² is selected from the groupconsisting of (6-10C)aryl and (1-9C)heteroaryl, wherein both groupsoptionally can be substituted; R³ is selected from the group consistingof: (1-6C)alkyl, —(CH₂)n-(3-7C) heterocycloalkyl),—(CH₂)n-(4-8C)heterocycloalkenyl), (3-7C)heterocycloalkyl, (6-10C)aryl,(1-9C)heteroaryl, —(CH₂)_(n)-(6-10C)aryl, —O-(6-10C)aryl, —C(═O)N, andcyano, wherein the groups can be substituted and wherein n is an integerof 0, 1 or
 2. 10. The method according to claim 1, wherein the TTKinhibitor is a chemical compound belonging to the class of compoundsaccording to Formula IV:

wherein, R¹ is selected from the group consisting of (3-6C)cycloalkyl,(3-7C)heterocycloalkyl, wherein the groups optionally can besubstituted; R² is selected from the group consisting of (6-10C)aryl and(1-5C)heteroaryl, wherein both groups optionally can be substituted. 11.The method according to claim 1, wherein the TTK inhibitor is a chemicalcompound belonging to the class of compounds according to Formula V:

wherein, R¹ is selected from the group consisting of hydrogen,(1-6C)alkyl, halo(1-6C)alkyl, HO(1-6C)alkyl, (3-6C)cycloalkyl,(3-7C)heterocycloalkyl or (1-5C)heteroaryl, wherein the groupsoptionally can be substituted; R² is (6-10C)aryl or (1-9C)heteroaryl,wherein the groups optionally can be substituted; R³ is selected fromthe group consisting of X-(6-10C)aryl and X-(1-9C)heteroaryl, whereinboth groups optionally can be substituted, wherein X representsS(═O)_(p), O, NR⁴, CR4aR4b, C═CR^(4a)R^(4b), wherein p is an integer of0, 1, 2 and further wherein R⁴, R^(4a), R^(4b) represent independentlyfrom each other a hydrogen atom or (1-6C)alkyl.
 12. The method accordingto claim 1, wherein the TTK inhibitor is a chemical compound belongingto the class of compounds according to Formula VI:

wherein, R¹ represents a phenyl group, a pyridyl group or an indolylgroup wherein the groups can optionally be substituted; R² represents aphenyl group, a pyridyl group or a pyrimidyl group wherein the groupscan optionally substituted; R³ represents a group selected from ahydrogen atom or —C(═O)—O—(CR⁷R⁸)—O—C(═O)—R⁴, wherein R⁴ represents agroup selected from: (1-6C)alkyl, substituted one or more times,identically or differentially, with a group selected from: —NH₂,—N(H)R⁵, —N(R⁵)R⁶, (4-7C)heterocycloalkyl, optionally substituted, oneor more times, identically or differentially, with a group selected from—NH₂, —N(H)R⁵, —N(R⁵)R⁶; R⁵ and R⁶, independently from each other,represent a group selected from a hydrogen atom and (1-3C)alkyl; R⁷represents a group selected from a hydrogen atom and (1-3C)alkyl; R⁸represents a hydrogen atom.
 13. The method according to claim 1, whereinthe TTK inhibitor is a chemical compound belonging to the class ofcompounds according to Formula VII:

wherein, R¹ is selected from the group consisting of (6-10C)aryl,wherein the group optionally can be substituted; R² is selected from thegroup consisting of (6-10C)aryl, wherein the group optionally can besubstituted.
 14. The method according to claim 1, wherein the TTKinhibitor is a chemical compound belonging to the class of compoundsaccording to Formula VIII:

wherein, R¹ is selected from the group consisting of hydrogen atom oramino; R² is selected from the group consisting of (6-10C)aryl,(1-5C)heteroaryl, (1-6C)alkyl, (3-6C)cycloalkyl and(3-7C)heterocycloalkyl, wherein the groups optionally can besubstituted; R³ is selected from the group consisting of (6-10C)aryl,wherein the groups optionally can be substituted; X is C or N.
 15. Amethod to determine whether a chemical compound is a TTK inhibitor, themethod comprising the steps of: a) Providing first and second mammaliancell lines, wherein the first cell line is CTNNB1-mutated and the secondcell line is CTNNB1 proficient; b) Contacting the first and second celllines with a first candidate compound; and, c) Determining by assay theinhibition of cell proliferation of the first and second cell lines. 16.The method according to claim 15, wherein steps b) and c) are repeatedwith a second candidate compound and a selection of candidate compoundis made based on the activity of the respective candidate compounds inthe assay with the first cell line.
 17. The method according to claim15, wherein the first and second cell lines are cancer cell lines. 18.The method according to claim 15, wherein the first and second celllines are isogenic cell lines.